CZ283478B6 - Recombinant dna molecule capable of encoding icam-3 or functional derivative thereof, processes for obtaining thereof and use - Google Patents

Abstract

Intercellular Binding Molecules (ICAM-3), which are involved in the processes by which leukocytes recognize and migrate inflammatory sites and connect to cell substrates during inflammation. Said molecules, screening assays are described to identify these molecules and antibodies capable of binding to these molecules. Also described is the therapeutic and diagnostic use of adhesion molecules and antibodies capable of binding thereto

Description

Technical field

The invention is based on the discovery of a new intercellular adhesion molecule, called ICAM-3, which participates in processes that recognize leukocyte populations and adhere to cell substrates. ICAM-3 mediates cellular interactions with other lymphocytes, macrophages and neutrophils at inflammatory and immune response sites.

ICAM-3, alone or in combination with ICAM-1 and / or ICAM-2, is useful for inhibiting intercellular adhesion of granulocyte, lymphocyte or macrophage family cells. The use of these molecules enables a method of treating specific and non-specific inflammatory diseases. The invention is further applicable to therapeutic and prophylactic methods of controlling HIV leukocyte infection, particularly HIV-1, in an individual exposed to or infected with HIV by administering ICAM-3, alone or in combination with ICAM-1 and / or ICAM-2. It thus enables the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) which are caused by the HIV virus. The invention is also applicable to therapeutic methods for 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 allows the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) caused by HIV-1. It is further useful for therapeutic methods of 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 allows the use of ICAM-3, alone or in combination with ICAM-1 and / or ICAM-2, for the treatment of asthma.

The invention further relates to molecules capable of binding to ICAM-3 (hereinafter referred to as antiICAM-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 allows the use of anti-ICAM-3 molecules, alone or in combination with santi-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. These are therapeutic and prophylactic methods of controlling HIV leukocyte infection, particularly HIV-1, in an individual exposed to or infected with HIV by administering anti-ICAM-3 alone or in combination with santi-ICAM-1 and / or anti-ICAM -2. It thus allows the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) caused by HIV. Further, they are therapeutic methods of inhibiting the migration of HIV-1 infected cells from the circulatory system using an antiICAM-1 composition, alone or in combination with an anti-ICAM-1 and / or antiICAM-2 composition. It thus enables the treatment of diseases such as AIDS (acquired immunodeficiency syndrome) caused by HIV-1.

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

BACKGROUND OF THE INVENTION

A. Attachment and function of leukocytes

Leukocytes must be able to attach to cellular substrates to properly protect hosts against foreign invaders such as bacteria or viruses, for a summary of these functions, see Eisen, HW (Microbiology, 3rd Ed., Harper & Row, Philadelphia, PA (1980), p. 290-295 and 381-418). Leukocytes must be able to attach to endothelial cells in order to migrate from the circulatory system to sites of inflammation. In addition, they must be coupled to antigen-presenting cells for a normal immune response to proceed, and finally, they must be coupled to appropriate target cells in order for the virally infected or tumor cells to decompose.

Recently, leukocyte surface molecules involved in mediating said fusion-mediated functions have been identified using hybridoma technology. Briefly, monoclonal antibodies directed 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 leukocyte surfaces and inhibited the fusion-related functions described above (Springer, T. et al., Fed. Proc. 44, 2660-2663). (1985)). The molecules identified by these antibodies have been termed Mac-1 and LFA-1 (Lymphocyte Function-associated Antigen-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granule 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 cell adhesion (Keizer, G. et al., Eur. J. Immunol. 15, 1142-1147 (1985)) .

The above leukocyte molecules have been found to be members of a related 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 having a single α chain and one β chain. While the chain a of each antigen differed from the others, the β chain was found to be highly conserved (Sanchez-Madrid, F. ad., J. Exper. Med. 158, 1785-1803 (1983)). The β chain of the glycoprotein family (sometimes referred to as CD 18) has been found to have a molecular weight of 95 kd, while the α chains range from 150 kd to 180 kd (Springer, T., Fed, why. 44, 2660-2663 (1985)) . Although the subunits and membrane proteins do not share the high homology shared 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. Copper. 158: 586-602 (1983); J. Exper. Copper. 158, 1785-1803 (1983)).

A group of individuals have been identified that are unable to express normal amounts 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 have been 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 defect manner in vitro as their normal counterparts in the presence of anti-CD18

B6 monoclonal antibodies. That is, they are unable to perform adhesion-related functions such as aggregation or attachment to endothelial cells. More importantly, however, these patients are unable to exert a normal inflammatory response due to the inability of their granulocytes to attach to cellular substrates. Very noteworthy is the finding that the 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 connection is an essential step in extravasation.

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 cell substrates (Anderson, DC et al., Fed. Proc., 44, 2671-2677 (1985), Anderson, DC et al., J. Infect. Dis 152: 668-689 (1985)). These facts show that if lymphocytes are incapable of adhesion in the normal way due to a lack of functional CD-18 family adhesion molecules, immune responses are inhibited.

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 leukocyte adhesion to other leukocytes, endothelial cells and other non-vascular cells. It has been found that the LFA-1, Mac-1 and p150, 95 surface receptor molecules are very closely related. Human subjects lacking these surface receptor molecules in leukocytes are subject to chronic and recurrent infections and exhibit other clinical symptoms, including defective drug responses.

Furthermore, since leukocyte adhesion is involved in the process by which foreign tissue is identified and rejected, 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. Viruses have a similar structure and encode proteins with a similar function. HIV infection is thought to occur through the binding of a viral 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 during this process destroys the T cell. The direct consequence of HIV infection is therefore the destruction of the individual's T4 population.

The destruction of T cells deteriorates the ability of the infected patient to prevent accidental infections. Although individuals with AIDS often develop cancer, the relationship between these cancers and HIV infection is in most cases uncertain.

Although mere HIV replication for the infected cells is lethal, such replication is most often detected only in a small fraction of the T4 cells of the infected individual, recent results show that there is a greater extent of viremia than originally thought, and the frequency of T cell infection can reach up to 1%.

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

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In addition to HIV replication, cells in HIV infection can be killed by cytotoxic murder cells. The killing cells are normally present in the human body and serve to monitor the host and destroy any foreign cells (for example, in inappropriate blood transfusion or organ transplant, etc.) 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 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 acquire the appearance of 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 by which HTV may cause T4 cell death and which is of particular interest 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. By HTV infection, infected cells will acquire the ability to blend with other T4 cells, both HIV-infected and uninfected healthy cells. Syncytium cannot function and soon perishes. Its destruction thus destroys 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 gain the ability to blend in with healthy cells.

HIV infection, and in particular HIV-1 infection, appears to affect the cell surface expression of leukocyte integrins and cellular adhesion responses mediated by these heterodimers (Petit, AJ et al., J. Clin. Invest. 79, 188 (1987), Hildreth, JEK et al., Science 244, 1075 (1989), Valentin, 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. ad., 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, which can be suppressed by treating infected cells with anti-CD18 or anti-CD11a monoclonal antibodies or by endothelial substrates act with anti-ICAM-1 antibodies (Rossen, RD et al., Trans. Assoc. American Physicians 102, 117-130 (1989)). Monoclonal antibodies to CD18 or CD11a have also been found to be able to inhibit the formation of syncytia involving phytohemagglutinin (PHA) stimulated lymphoblastoid cells and constitutively infected CD4-negative T cells (Hildreth, J. E. K. et al., Science 244, 1075 (1989)). It has been shown that the action of anti-CD18 or anti-CD11a monoclonal antibodies only on virus-infected cells has little effect on syncytia, leading to the suggestion that these antibodies protect mainly uninfected target cells from infection (Hildreth, JEK et al., Science 244 , 1075 (1989), Valentin A.A., J. Immunology 144, 934-937 (1990)). Valentin ad. (Valentin, A. et al., J. Immunology 144, 934-937 (1990)) have recently confirmed these findings 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.

Although the mechanism by which monoclonal antibodies specific for CD18 or CD11a protects endangered cells from merging with HIV-infected cells remains unknown and is not necessary to understand the invention, studies with radiolabeled gp120 indicate that CD18-containing heterodimers do not provide a binding site for virus (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 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.

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Thus, the invention is in part 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 HTV infected T cells to adhere to 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 resting T cells. Only anti-ICAM-3 antibodies are able to block T cell adhesion to LFA-1 when T cells are not 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 that allows the transfer of HTV-1 from the infected cell to a healthy cell of an individual 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 virus-infected leukocytes. Of particular importance is the migration and spread of HIV-infected leukocytes. Migration of such cells creates extravascular foci that 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 infiltrates in the central nervous system 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), H. 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 yet been precisely defined. These mechanisms may include either the transport of virus, free of cells, or the transport of virus across endothelial barriers in the cytoplasm of infected mononuclear cells.

Because HIV-1 infection stimulates cell surface expression of molecules that allow the adhesion of leukocytes 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)), use antibodies that inhibit cell migration (WO 90/13316) to prevent the spread of HIV-infected cells.

D. Asthma - clinical characteristics

Asthma is a heterogeneous group of diseases. It is characterized by irritable tracheobronch hyperreactivity (Kay, AB, Allergy and Inflammation, Academic Press, NY (1987). Clinically, asthma is characterized by intense narrowing of tracheobronch, dense viscous secretions, dyspnoea, coughing and shortness of breath. however, the net result is increased resistance to airflow, excessive lung and thorax filling, and abnormal distribution of oxygenation and blood flow to the lungs.The disease is manifested by episodic periods of acute symptoms alternating between periods of asymptomatic acute episodes leading to hypoxia and may be fatal. affects about 3% of the world's population.

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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 reactions such as bruising and inflammation for intradermal injection of airborne antigens (such as pollen, environmental and occupational contamination, etc.) and elevated levels. IgE in serum. 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 IgE responses, controlled by T and B lymphocytes, and activated by the interaction of airborne antigen with pre-formed IgE molecules bound to a living cell. The antigens must meet 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 inducing antigen, environmental factors, occupational factors, physical exertion and emotional stress.

Asthma can be treated with methylxanthines (such as theophylline), beta-adrenergic agonists (such as catecholamines, resorcinols, saligenins and ephedrine), glucocorticoids (such as hydrocortisone), inhibitors of mast cell degranulation (such as chromones such as sodium cromolyn) and anticholinergics (such as atropine).

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

Studies of washed bronchoalveol (Godard, P. et al., J. Allergy Clin. Immunol. 70, 88 (1982)) and epithelial depleted respiratory smooth tissue tissue have allowed a deeper insight into the immunological base of asthma (Flavahan, NA et al., J. Appl. Physiol., 58, 834 (1985), Bames, PJ et al., Br. J. Pharmacol., 86, 685 (1985)). Although these studies did not elucidate the mechanism of asthma immunology, they have led to the development of a generally accepted hypothesis regarding the immunological etiology of the disease (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 of eosinophils and destruction of mucociliary capacity. According to the eosinophilic hypothesis, eosinophils are attracted to bronchus to neutralize harmful mediators released by living lung cells, according to this hypothesis, eosinophils are attracted to bronchi where they degrade and release cytotoxic molecules. In degranulation, eosinophils release enzymes such as histaminase, arylsulfatase and phospholipase D, which enzymatically neutralize harmful mediators of the living cell. At the same time, these molecules promote the destruction of the mucociliary apparatus, preventing the cleansing of the bronchial secretion and contributing to asthma-specific lung damage.

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

SUMMARY OF THE INVENTION

The subject of the invention is:

a recombinant DNA molecule capable of encoding ICAM-3 or a functional derivative thereof, wherein said DNA molecule comprises at least one fragment selected from the group consisting of nucleotide sequences corresponding to SEQ ID NOS: 1, 3 and 5, and further particularly capable of expressing in ICAM-3 cell or a functional derivative thereof,

A functional derivative of ICAM-3, wherein said derivative is an ICAM-3 fragment selected from the group consisting of amino acid sequences corresponding to sequence numbers 2, 4 and 6, particularly wherein said fragment is selected from the group consisting of D1, D1-2 , D1-3, D1-4 and D1-5 ICAM-3,

ICAM-3 or a functional derivative thereof, obtained by expressing a recombinant DNA molecule in a cell, a method of producing ICAM-3 or a functional derivative thereof, comprising expressing a recombinant DNA molecule in a cell, an antibody or antibody fragment capable of binding to a molecule selected from ICAM -3 and a functional derivative of ICAM-3, particularly wherein binding of said antibody to said molecule reduces the ability of said molecule to bind to a receptor molecule, particularly wherein the receptor is LFA-1, MAC-1, or p150.95.

The present invention also provides a method of 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) fusing the spleen cells of an animal with a myeloma cell line,

(c) allowing fused 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.

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

(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 said extract of said cell, which method is performed in vitro.

The present invention further provides a method for diagnosing the presence of ICAM-3 in a sample of a fluid taken from a mammalian subject, comprising:

(a) incubating the biological fluid sample of said subject with a composition comprising an antibody or fragment thereof capable of binding to a cell expressing ICAM-3; and

b) detecting said antibody bound to said fluid, the method being carried out in vitro. In particular, said fluid is selected from the group consisting of blood, serum, plasma, synovial fluid, amniotic fluid, spinal fluid, or urine.

The invention further provides a method for identifying agents capable of modulating the binding of IC AM3 to LFA-1 by contacting ICAM-3 with LFA-1 in the presence of such agent and measuring said modulation of ICAM-3 / LFA-1 binding. . In particular, the method is performed either by LFA-1 and ICAM-3 being purified to remove naturally occurring impurities, or LFA-1 is expressed on the cell surface and ICAM-3 is purified to remove naturally occurring impurities

Or ICAM-3 is expressed on the cell surface and LFA-1 is purified to remove naturally occurring impurities.

The invention further provides a pharmaceutical composition for modulating the biological functions of a cell mediated by ICAM-3, comprising a composition selected from the group consisting of an antibody capable of binding to ICAM-3, a fragment of such antibody capable of binding to ICAM-3, ICAM-3 -3 and a non-immunoglobulin ICAM-3 antagonist other than ICAM-1, ICAM-2, or a member of the CD-18 family. The composition preferably further comprises an immunosuppressive agent.

The invention further provides a pharmaceutical composition comprising an ICAM3 modulating agent in combination with a pharmaceutically acceptable carrier, in particular in combination with at least one other immunosuppressive agent.

The present invention also provides a therapeutic composition comprising an antibody molecule or fragment thereof in combination with a pharmaceutically acceptable diluent, vehicle or carrier.

The invention further provides a diagnostic composition comprising the antibody molecule or fragment thereof in a detectably labeled form.

Overview of the drawings

Giant. 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 MAFAs specific for LFA-1, ICAM-1, ICAM-2, and ICAM-3. Control wells were coated without LFA-1.

Giant. 2. Flow cytometric analysis of cell 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 controls 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.

Giant. 3. Immunoprecipitation of ICAM-3. (A) ¹²⁵ I-labeled cell lysates immunoprecipitated with non-X63 MAb CB-IC3 / 1 (anti-ICAM-3) non-binding control. (B) ¹²⁵ I-labeled SKW3 cell lysates, treated with (+) or without (-) N-glycanase and immunoprecipitated with non-binding control MAb X63, with MAb W6 / 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).

Giant. 4. Immunoprecipitation of ICAM-3 from various sources. ICAM-3 was immunoprecipitated from ¹¹²⁵ labeled lymphocyte and neutrophil lysates by mAb-coupled Sepharose. Immunoprecipitated ICAM-3 was digested by polyacrylamide gel electrophoresis. 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).

Giant. 5. Effect of PMA on SKW3 binding to ICAM-1 and ICAM-3. The ability of SKW3 cells to bind to purified ICAM-1 and ICAM-3 and the effect of PMA stimulation on this binding was tested as previously described (Dustin et al., Nature 341, 619 (1989), Mariin et al., Cell 51, 813-819 ( 1987)). One representative experiment is shown and error bars indicate one standard deviation (SD).

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Giant. 6. Blocking of PMA stimulated SKW3 cell adhesion by antibodies. Various antibodies were tested for their ability to block the binding of PMA stimulated SK.W3 cells to purified ICAM-1 or ICAM-3 drive as described (Dustin et al., Nature 341, 619 (1989), Mariin et al., Cell 51, 813-819). (1987)). One representative experiment is shown and error bars show one SD.

Giant. 7. Binding of COS cell transfectants to purified ICAM. COS cells were transfected with the LFA-1 drive expression vector as described (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 show one SD.

Giant. 8. Temperature dependence of PMA stimulated SK.W3 binding to ICAM. The ability of PMA-stimulated SKW3 cells to bind purified ICAM-1 and ICAM-3 was tested at 4, 16, 22 and 37 ° C as described previously (Mariin 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 show one SD.

Giant. 9. Blocking the division of PHA-stimulated cells by T antibodies. Various antibodies were tested for their ability to block PMA-stimulated division of T cells as previously described (Krensky et al., J. Immunol. 131, 611-616 (1983)).

Giant. 10. Influence of anti-ICAM antibodies on mixed lymphocyte reaction. Various antibodies have been tested for the ability to block a mixed lymphocyte response in the manner described (Krensky et al., J. Immunol. 131, 611-616 (1983)).

Giant. 11. Gas chromatogram of peptide fragments of ICAM-3. ICAM-3 were purified and digested with Lys-C. Peptide fragments were distinguished by high performance liquid chromatography.

Giant. 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.

Giant. 13. Sequence comparison of NK-10-initiated sequence with human ICAM-1. The GAP program was used to identify sequence homology of the NK-10-initiated sequence and human ICAM-1 protein.

Giant. 14. Sequence comparison of the RM13-initiated sequence with human ICAM-1. The GAP program was used to identify sequence homology of the RM13 initiated sequence with human ICAM-1.

Giant. 15. Sequence comparison of the RM13-initiated sequence with human ICAM-2. The GAP program was used to identify sequence homology of the RM13-initiated sequence and human ICAM-2.

Giant. 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.

Giant. 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.

Giant. 18. ICAM-3 partial sequence. The DNA sequence of clone 11.2 was determined by standard procedures.

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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 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, which are involved in the cell 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, agranulocyte natural killer cells 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 newly identified ICAM-3 (according to the invention). These surface molecules are constitutively expressed on some tissues and inflammation induced on others (Mariin, 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 Sep. 28, 1988).

LFA-1 functions in both antigen-specific and antigen-independent T-cytotoxic interactions. 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 print)).

II. ICAM-1

ICAM-1 is a single chain glycoprotein whose mass ranges 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 et al., Cell 52, 925-933 (1988), Simmons, D. et al., Nature 331, 624-627 (1988)). ICAM-1 can be via cytokines including IFN-γ. TNF and IL-1 were introduced on cells of various types (Dustin, M.L. et al., Immunol. Today 9, 213-215 (1988)). Introduction of ICAM-1 on epithelial cells, endothelial cells and afibroblasts mediate LFA-1 dependent lymphocyte adhesion (Dustin, ML et al., J. Immunol. 137, 245-254 (1986); Dustin, ML et al., J. Cell. Biol. 107: 321-331 (1988); Dustin, ML et al., J. Exp. Med. 167, 1323-1340 (1988)). Adhesion is blocked by pretreatment of LFA-1 MAb lymphocytes or pretreatment of a second ICAM-1 MAb cell (Dustin, ML et al., J. Immunol. 137, 245-254 (1986); Dustin, ML et al., J. Cell. Biol. 107, 321-331 (1988), Dustin, ML et al., J. Exp Med., 167, 13231340 (1988)). Identical results with purified ICAM-1 on synthetic membranes or Petri dishes demonstrate that LFA-1 and ICAM-1 are receptors of each other (Mariin, SD et al., Cell 51, 813-819 (1987), Makgoba, MW 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.

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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, MW 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 by cell distribution and the absence of cytokine induction. ICAM-2 is an integral membrane protein with 2 Ig-like regions, while ICAM-1 has 5 Ig-like regions (Staunton, D.

E. et al., Cell 52, 925-933 (1988), Simmons, D. et al., Nature 331, 624-627 (1988). Surprisingly, ICAM-2 is much closer related to the two most N-terminal regions of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 to other members of the Ig superfamily, thus demonstrating the subfamily of Ig ligands that bind to receptors from families of the same integrins. Further data on ICAM-2 can be found in US Patent Application No. 07 / 454,294.

IV. ICAM-3

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

The development of MAb to ICAM-2 has allowed the analysis of many formerly 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 cell SK.W3 lymphoma cell line (FIG. 1) there was a pathway for adhesion to LFA1 independent of ICAM-1, ICAM-2.

To characterize this adhesion pathway, the MAbs were immunized on SK.W3 and screened for the ability to inhibit binding of this cell line to purified LFA-1 in accordance with anti-ICAM-1 and anti-ICAM-2 MAbs. One MAb, CBR-IC3 / 1, was chosen to completely inhibit this new adhesion pathway (Fig. 1). SKW3 adhesion to purified LFA-1 was only slightly inhibited by the combination of blocking antiICAM-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 anti-ICAM-2 MAb. Thus, SKW3 adhesion to purified LFA-1 was strongly mediated by ICAM-3 and also partially by ICAM-2. In adhesion to LFA-1, each of the four cell lines utilized these three ICAMs to varying degrees, as demonstrated by different MAb inhibition patterns. The adhesion of the lymphoblastoid cell line B, JY, was first via 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, since adhesion was not inhibited by the MAbs by either ICAM-1 or ICAM-3 alone, but almost completely by both MAbs together. Thymoma cell line. Jurkat, used ICAM-2 and ICAM-3 pathways with little ICAM-1 contribution. The adhesion of T lymphocytes, both resting and activated by phytohemagglutinin (PHA, Fig. 18) was also investigated. Resting lymphocytes have previously shown 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, expression of ICAM-1 was induced and adhesion to LFA-1 proceeded mainly through 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 of the MAbs. A notable exception is the resting lymphocyte adhesion to LFA-1, which was 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 measured by

By immuno fluorescence cytometry (Fig. 2). Comparing the surface expression of ICAM and their contribution to LFA-1 adhesion, it was found that ICAM-1 has the greatest affinity for LFA-1, while ICAM-2 and ICAM-3 have affinities similar but lower. For example, Jurkat cells expressed ICAM2 and ICAM-3 at a similar level and both contributed to binding to LFA-1. When ICAM-2 expression was greater than ICAM-3, such as JY cells, ICAM-2 pathway over ICAM-3 predominated in adhesion to LFA-1. In contrast, SLA and SK.W3 expressed three-fold more ICAM-3 than ICAM-2, and not surprisingly the ICAM-3 pathway in adhesion outweighed the ICAM-2 pathway.

On resting lymphocytes, ICAM-3 was well expressed and ICAM-1 was absent, adhesion to LFA-1 was mainly mediated 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 ICAM-3 distribution pattern 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 the cell lines was consistent with the LFA-1 dependent pathway independent of ICAM-1, ICAM-2. Cell lines (HUVEC, Paradise), binding only LFA-1 through 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 of adhesion had correspondingly low surface expression of ICAM-3 (data not shown).

ICAM-3 differed significantly in leukocyte expression from ICAM-1 and ICAM-2 (Fig. 28). ICAM-3 was expressed at high levels on resting lymphocytes, monocytes and neutrophils, while ICAM-1 and ICAM-2 were expressed much weaker or absent. In comparison, ICAM-1 and ICAM-2 were poorly expressed on monocytes and only ICAM-2 was present on resting lymphocytes. Neither ICAM-1 nor ICAM-2 was expressed on neutrophils. Following activation of PHA lymphocytes, ICAM-3 expression increased 2-3-fold, while ICAM-1 expression was strongly stimulated (Dustin et al., J. Immunol. 137, 245-254 (1986); Figure 2B).

ICAM-3 immunoprecipitates from various ¹²⁵ 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 previously described adhesion molecules, including human leader receptors LAM-1 (Tedder et al., J. Immunol. 144, 532-540 (1990)), inducible endothelial adhesion molecules. VACAM-1 (Rice et al., J. Exp. Med. 171, 1369-1374 (1990, Carlos et al., Blood, in press (1990), Osbom et al. Cell 59, 1203-1211 (1989)) and the matrix family. VLA receptors (Hemler, E., Ann. Rev. Immunol. 8, 365-400 (1990)) and in the databases of the fourth leukocyte workshop (Gilks, WR et al., Leukocyte Typing Data Base IV, Oxford University Press, Oxford, England (1990)), MAbs with similar cell distribution were 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 (Wavryk et al., Immunol. Rev. 108, 135-161 (1989)) ). Because 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 through tissue endothelium (Hamann et al., J. Immunol. 140, 693-699 (1988)). Mackay et al., J. Exp. Med 171: 810-817 (1990), Pals et al., J. Immunol., 140, 1851-1853 (1988), and Nunoi et al., Hum Path. 759 (1988)). The function of ICAM-3 on neutrophils remains

Unclear to date, 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. d., Scand J. Immunol., 22, 619-631 (1985)). The finding that adhesion of resting T lymphocytes to LFA1 occurs primarily through ICAM-3, coupled with the fact that ICAM-3 is much better expressed than other LFA-1 ligands on monocytes and resting lymphocytes, points to an important role in initiating immune responses. 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)). ICAM-3 may play a certain role as such, particularly on resting T cells where ICAM-1 and ICAM-2 are not well expressed. Indeed, a role is 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, the importance of ICAM-3 in antigen-specific interactions between T and B lymphocytes can be expected to be important if one of these cells has not yet been activated. ICAM-3 may also be important in the breakdown of some targets by T cells, which is dependent on LFA-1 but not ICAM-1 (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 extending the life of renal (Cosimi et al., J. Immunol. 144, 4604-4612 (1990)) acardial (Flavin et al., Transplant. Proc., 23, 533534 (1991)) allotransplants. MAb ICAM-3 inhibits a characteristic and overlapping number of immune responses in vivo, since it inhibits the 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 that contains the ICAM-3 gene. Such analysis can be performed by transfecting the 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 adhesive molecules. According to this method, a cDNA library is prepared from cells that express ICAM-3 (such as lymphoblastoid cell lines SLA, Jurkat or SKW3). Preferably, the cDNA library is 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. Transfected cells containing ICAM-3 encoding sequences expressing this ligand on their cell surface are adhered to the surface of the Petri dish to LFA-1 or anti-ICAM-3 antibodies. Non-adherent cells are removed by washing, adherent cells are removed from the Petri dish and cultured. The sequences in these cells expressing recombinant ICAM-3 are then determined.

When LFA-1 is used to cover the Petri dish, specific anti-ICAM-1 and anti-ICAM-2 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, ie MAb anti-ICAM-1, and CBR-IC2 / 2, ie MAb anti-ICAM -2. Binding of ICAM-3 transfected cells to LFA-1 coated petri dishes is inhibited by EDTA and anti-LFA-1 MAb but not inhibited by anti-ICAM-1 or anti-ICAM- MAb

2. Therefore, cells expressing ICAM-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.

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One method of 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 codon preference combinations, homology to known ICAM-1 or ICAM-2 DNA sequences, and codon degeneration is used to construct probes. The 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 cloning of human aldehyde dehydrogenase genes (Hsu, LC et al., Proc. Natl. Acad. Sci. USA 82, 3771-3775 (1985)), fibronectin (Suzuki, S. et al., Eur. Mol. Biol. Organ. J. 4, 2519-2524 (1985)), the human estrogen receptor gene (Walter, P. ad., Proc. Natl. Acad. Sci. USA 82, 7889-7893 (1985)) and plasminogen activator. tissue type (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 reactions of cDNA or mRNA from cells known to express an ICAM-3 protein such as SKW3 or tonsila (deFougerolles et al., J. Exp Med 175: 185-190 (1992)). 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 expression of full-length clones, for example in COS cells, and by testing for reactivity with mAb ICAM-3.

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

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.

The term ICAM-3 fragment refers to any polypeptide subunit of a molecule. Particularly preferred are ICAM-3 fragments having ICAM-3 activity and that are soluble (i.e., do not bind to membranes). The soluble fragment is preferably formed by draining the membrane

Corresponding regions of the parent molecule or by deletion or substitution of hydrophobic amino acid residues by hydrophilic residues. 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, D13, 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 when one of the molecules contains a structure that is not present in the other molecule or if the sequence of amino acid residues 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 the solubility, absorption capacity, half-life, etc. of the molecule. Alternatively, these units may reduce the toxicity of the molecule or eliminate or attenuate any undesirable 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. The 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 proximity of the cell and thus promotes cell death. Any suitable toxin unit may be used; however, it is preferable 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 by reacting a target amino acid residue 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 agents can be used to form cross-links of ICAM-3 with a water-insoluble carrier matrix. Alternatively, reactive water-insoluble matrices 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 can be used to generate the final derivative as long as the final derivative has the desired efficacy. Obviously, a mutation made in a DNA encoding a functional derivative must not get a sequence outside the reading frame and preferably should not create complementary regions that could produce a secondary structure of mRNA (see published patent application EP 75 444).

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At the genetic level, functional derivatives may be prepared by mutagenesis of site-directed ICAM-3 encoding DNA to produce DNA encoding a functional derivative and subsequent 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 that can then be expressed and screened for the optimal combination of 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 derivative of ICAM-3 according to the invention is preferably carried out by site-directed mutagenesis. 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 ICAM-3 derivatives using specific oligonucleotide sequences that encode the DNA sequence of the desired mutation.

Amino acid deletions, insertions, or substitutions may be accomplished using site-directed mutagenesis. Deletion of amino acid sequences usually comprise about 1 to 30 residues, preferably 1 to 10 residues, and most commonly are contiguous. Most preferred 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 single or fused amino acid residues in sequences encoding ICAM-3 as well as terminal condensations with polypeptides of substantially unlimited length. Internal insertions (i.e. insertions within the complete sequence of an ICAM-3 molecule) can generally comprise about 1 to 10, preferably 1 to 5 residues. An example of 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 made when fine modulation of the characteristics of the ICAM-3 molecule is desired, in accordance with this table.

Table 1

the original rest For example, substitution Ala giy, ser Arg lys Asn gin, his Asp glu Cys ser Gin asn Glu asp Gly ala, for His asn, gin Ile leu, wall Leu ile, wall Lys arg, gin, glu

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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 their effect on maintaining (a) the structure of the polypeptide chain in the region of substitutions such as flame or spiral conformations. (b) the 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) the electropositive side chain residue replaces (or is replaced by) an electronegative charged residue; (c) the bulky side chain residue replaces a residue that does not have such a side chain (or is replaced by 20).

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

Mutations that increase the affinity of ICAM-3 can be made by introducing amino acid residues that are present at homologous positions in ICAM-1 or ICAM-2. Similarly, mutated ICAM-3 molecules lacking the N-linked CHO moiety at ICAM-1 or ICAM-2 homology positions can be prepared.

It is difficult to predict the exact effect that particular substitutions, deletions, or insertions will have on the biological activity of ICAM-3. However, the person skilled in the art is aware that this effect can be ascertained by routine screening tests. For example, a derivative by mutagenesis is generated by scanning a DNA linker encoding a native ICAM-3 molecule. The derivative is then expressed in a recombinant host and optionally purified from cell culture, for example, by immunoaffinity 35 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 protein properties such as redox or thermal stability, half-life, susceptibility to proteolytic degradation, or 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 its 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 prevents the ability of ICAM-3 to perform any of its biological functions. Examples of such antagonists include ICAM-1 and ICAM-2, a functional derivative of ICAM-1 and ICAM-2, an anti-ICAM-3 antibody, an antiLFA-1 antibody, etc.

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Cell aggregation assays described in US Patent Application Nos. 07/045963, 07/115798, 07/155943, 07/189815 or 07/250446 can measure LFA-1 dependent aggregation and can therefore be used to identify agents that affect the extent of aggregation of ICAM-3 / LFA-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. Further, the assay can determine 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 PMA-stimulated peripheral T cells. Thus, the binding of peripheral blood cells to LFA-1 is used to test ICAM-3-mediated aggregation.

C. Anti-ICAM-3 antibody

A preferred immunoglobulin antagonist of the invention is an antibody to ICAM-3, such as CBR-IC3 / 1 described herein. Other suitable anti-ICAM-3 antibodies can be obtained by a variety of methods.

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 procedures known in the art or by the procedures described in PCT Patent Application Nos. PCT / US91 / 02942 and PCT / US91 / 02946, filed at a site of acceptance 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 sepitope (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 surface of lymphocytes. Introduction of such cells into an appropriate animal, for example by intraperitoneal injection, produces 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 by the Selden method. R. F. (European Patent Publication No. 289034) or Selden, R. F. et al. (Science 236, 714718 (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 ICAM-3 by the animal.

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 as described above 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 screened to determine if they inhibit such aggregation by binding to ICAM-3 or to 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 those antibodies that bind to members of the CD-18 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 vice versa).

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The ability of the 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 compositions 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 ICAM3, but not to cells that do not express ICAM-3.

Other agents in addition to the above ICAM-3 agonists and antagonists that can be used in accordance with the invention in the treatment of inflammation, HTV 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 to 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.

Since the CD-18 family molecules are capable of binding to ICAM-3. administration of such molecules (for example, as heterodimers containing both α and β subunits, or as molecules composed solely of α and β subunits, or as molecules containing fragments of either or both subunits) will compete with cells for binding to ICAM-3 present on cell.

The anti-aggregation antibodies of the invention can be identified 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 US Patent Application Nos. 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 can be readily determined 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 capable of identifying and distinguishing between antibodies capable of binding to ICAM-3 versus members of the CD- 18.

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); -3, functional derivatives of ICAM3 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 different 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 above methods, processes or technologies to obtain a particular anti-inflammatory agent. and technology combine.

- 19 GB 283478 B6

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 is derived from the ability of ICAM-3 and its functional derivatives to interact with receptors of the CD-18 family of molecules, particularly LFA-1. Because ICAM-3 is able to interact with members of the CD-18 family of glycoproteins, it can be used to suppress (ie, prevent or control) inflammation.

The term inflammation includes both specific defense system reactions and non-specific defense system reactions.

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 inflammation is caused, mediated or associated with a specific defense system response. Examples of inflammation due to a specific defense system response include a response to antigens such as measles virus, autoimmune diseases, and delayed-type hypersensitivity responses mediated by T cells (as occurs, for example, in individuals having a positive Mantaux test). Other examples of specific immune system inflammatory responses are chronic inflammatory diseases and rejection of whole transplanted tissue and organs, such as kidney and marrow transplants.

A non-specific defense system reaction refers herein to a leukocyte-mediated reaction that is not capable of immunological memory. Such cells include granulocytes and macrophages. Inflammation herein is the result of a non-specific defense system response when the inflammation is caused, mediated or associated with a non-specific defense system response. Examples of inflammations 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 myocardial injury or other tissues, 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.

As mentioned above, the binding of ICAM-3 molecules to members of the CD-18 molecule family is central to cell adhesion. Through the adhesion process, lymphocytes are able to continuously monitor an animal for the presence of foreign antigens. Although 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), rejection of tissue transplants, and many autoimmune diseases. Therefore, any composition capable of inhibiting cellular 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 lymphocyte proliferation (Davignon, D. et al., Proc. Natl. Acad. Sci. USA 78, 4535-4539 (1981)) , antibody production (Fischer, A. ad., J. Immunol. 136, 3198-3203 (1986)) and effector functions of all leukocytes, such as the cytotoxic T cell lytic activity (Krensky,

-20GB 283478 B6

A. M. et al., J. Immunol. 132, 2180-2182 (1984)), macrophages (Strassman, G. ad., 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 the leukocyte to adhere to the respective cellular substrate, which then inhibits the biological function associated with binding. Such functions, as long as they involve 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 nephrotoxicity, that occur with conventional agents.

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, it can be used to suppress inflammation. In addition, functional derivatives and antagonists of ICAM-3 can be used to suppress inflammation.

1. Suppressors of delayed-type hypersensitivity reactions

ICAM-3 partially mediates the adhesion events 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 exploited in two ways. On the one hand, a composition comprising a monoclonal antibody to ICAM-3 or a soluble derivative of ICAM-3 may be administered to a patient with delayed-type hypersensitivity reactions. For example, such a composition may be administered to an individual in contact with antigens such as poison ivy, poison oak, and the like. In another embodiment, a monoclonal antibody capable of binding to ICAM-3 is administered to a patient in conjunction with an antigen to prevent subsequent inflammatory disease. reaction. Thus, additional administration of an antigen with an antibody to ICAM-3 may temporarily tolerate an individual against 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. Reynaud's syndrome, rheumatoid 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 the needy subject an anti-inflammatory agent in an amount sufficient to suppress 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

Non-immunoglobulin antagonist ICAM-3 other than LFA-1. Especially preferred are anti-inflammatory agents comprising 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 antibody to ICAM-1 and / or ICAM-2, or a fragment thereof.

The invention further encompasses the above methods of suppressing the inflammatory response of a specific defense system, further comprising administering to the subject an immunosuppressive agent. Such a composition is preferably administered at a lower (i.e., sub-optimal) dosage as 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 due to 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 a consequence of a non-specific defense system response when it is caused, mediated or associated with a non-specific defense system response. Examples of inflammations 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 trauma 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 agents 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, a non-immunoglobulin antagonist of ICAM-3 other than ICAM-1, ICAM-2, or a member of the CD-18 family of molecules.

B. Suppressors of organ and tissue rejection

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 reactions 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 tissue, for example, kidney, rejection of non-jawed 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

Immune responses 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 sickness. This situation can be solved by using the antibodies of the invention. In this embodiment of the invention, antibodies to ICAM-3 would be administered in combination with a therapeutic or diagnostic agent. The addition of antibodies will prevent 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 with antibodies capable of binding to LFA-1, may be used in the treatment of the disease. Thus, in a solubilized form, such molecules can be used to inhibit rejection reactions to organs or transplants. 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 may be used to inhibit metastasis of a hematopoietic tumor cell that requires a functional member of the CD-18 family to migrate. In accordance with this embodiment of the invention, a composition (such as an antibody capable of binding to ICAM3; toxin derivatizing such an antibody; an antibody fragment capable of binding to ICAM-3; toxin derivatizing said fragment; substantially pure ICAM-3; functional derivative of ICAM- 3 or a non-immunoglobulin antagonist (ICAM-3 other than ICAM-1 or ICAM-2) in an amount sufficient to suppress said metastasis.

Another embodiment of the invention provides a method of suppressing the growth of tumor cells expressing ICAM-3. Specifically, the method comprises administering the composition to a patient in an amount sufficient to inhibit this 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 family of molecules or a toxin-derivatized functional derivative of the member of the CD-18 family of molecules.

Another embodiment of the invention provides a method of suppressing the growth of a tumor cell expressing LFA1. Specifically, 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 HTV infection and preventing the spread of HTV infected cells

Another embodiment of the invention provides a method of controlling HTV infection. Specifically, the method comprises administering to an individual infected with HTV an effective amount of an HTV suppressant. Although the invention relates in particular to a method of controlling HTV-1 infection, it is to be understood that the method is applicable to any variant of HTV (such as HTV-2) that can infect cells in a manner that can be controlled by the compositions of the invention. . Such variants are HTV equivalents for purposes of the present 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 contact time of infected cells with uninfected cells. into uninfected cells. Compositions of the invention that are capable of modulating LFA-1 / ligand interactions can then suppress infection with HIV, particularly HTV-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

HIV-infected cells, bind to LFA-1 healthy T cells. ICAM3, an ICAM-3 fragment, a functional derivative of ICAM-3, or an antibody to ICAM-3 can 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 the dosage, route of administration, etc. of the composition is sufficient to control or prevent HIV infection. The compositions should be administered to patients who are at risk of 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 infection 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 further HTV infection.

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

The present invention provides improved AIDS therapy and an improved means of suppressing HIV infection, particularly HTV-1 infection, which comprises co-administering the following components:

(I) ICAM-3, soluble derivative of ICAM-3, CD11 (either CD11a or CD11b or CD11c), soluble derivative of CD11, 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.

Another application 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 an HIV-infected T cell 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 HIV (or other virus) infected cells. The amount is said to be sufficient to suppress T cell migration if the dosage, route of administration, etc. of the composition is sufficient to inhibit or prevent such migration.

These compounds can be used for either prophylactic or therapeutic purposes. When used prophylactically, the compositions of the invention are administered prior to discovery

Any symptom of a viral infection (for example, before, at or shortly after infection, but before any symptom of such infection). Prophylactic administration of the compositions serves to prevent or inhibit any subsequent migration of T cells. When used therapeutically, the composition is administered at (or shortly after) the detection of virally infected T cells. Therapeutic administration of the composition serves to inhibit 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 interactions is used in the treatment of asthma. Specifically, 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 suppress eosinophil migration by decreasing 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 reduce 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 aephedrine), glucocorticoids (such as hydrocortisone), chromones (such as is cromolyn sodium) 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 prophylactic 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 inhibit any subsequent asthmatic response. When used therapeutically, the composition is administered at (or shortly after) the onset of the asthma symptom. Therapeutic administration of the composition serves to attenuate any current asthmatic 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 labels (such as biotin, avidin, etc.), fluorescent labels, or paramagnetic atoms. Methods of such labeling are known. Labeled antibodies can then be used in diagnostic imaging. Clinical applications of antibodies in diagnostic imaging are summarized in Grossman, Η. B. Urol. Clin. North Amer. 13, 465-474 (1986)), Unger, E. C. et al. . Invest. Radiol. 20, 693700 (1985)) and Khaw, B.A. et al., Science 209, 295-297 (1980)).

The presence of ICAM-3 expression can also be detected using hybridization probes such as mRNA probes, cDNA probes, genomic DNA or synthetic oligonucleotides that bind to

ICAM-3 gene sequences or ICAM-3 mRNA sequences present in a cell that expresses ICAM-3. Methods for performing such hybridizations are described by Maniatis, T. et al., Molecular Cloning, and Laboratory Manual, Coldspring Harbor, NY (1982), and Haymes, BD et al., Nucleic Acid Hybridization, and Practical Approach, IRL Press, Washington, DC. (1985).

Detection of ICAM-3 expression foci using labeled antibodies or nucleic acid 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 assay can be used to monitor recipients of transplanted organs, e.g., kidney, for early signs of potential tissue rejection. These assays may also serve to determine the individual's prerequisites for creumatoid 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, Work, TS et al., North Holland Publishing Company, NY (1978), with particular reference to An Introduction to 20 Radioimmune Assay and Related Techniques, Chard, T. 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 generally 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 ICAM3 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, spinal fluid, or urine.

VIII. Administration of the compositions of the invention

The therapeutic effect of ICAM-3 can be achieved by administering to a patient an entire ICAM-3 molecule or any therapeutically effective peptide fragment thereof. Of particular interest are the 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 benefits of ICAM-3 can be enhanced by the use of functional derivatives of ICAM-3, in addition to additional amino acid residues, to improve carrier binding or to enhance ICAM-3 activity. The scope of the invention is further intended to include functional derivatives of ICAM-3 lacking certain amino acid residues when 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 preparations containing them 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 an animal or tissue culture or by recombinant DNA.

Administration of ICAM-3 or ICAM-3-derived molecules may be performed alone or in combination with ICAM-1 or ICAM-2. Administration of antibodies to ICAM-3 or other molecules capable of binding to ICAM-3 or ICAM-3-derived molecules may be performed

Alone or in combination with kICAM-1 antibodies and / or kICAM-2 antibodies. When administering antibodies or antibody fragments capable of binding to ICAM-3 to a patient or administering ICAM-3 (or a fragment, variant, or derivative thereof) to a patient, the dosage of the composition administered will vary depending upon factors such as the patient's age, weight, height, sex, general health, history of prior medical treatment, etc. It is generally desirable to provide the patient with an antibody dose in the range of about 1 µg / kg to 10mg / kg (patient 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 dosage which also ranges 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 kLFA-1, the antibody kICAM-1 and / or the antibody kICAM-2. Concomitant administration of two compounds herein means that both compounds are administered at such a short time interval that they can be detected simultaneously 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 or single or multiple boluses may be used.

The compositions of the invention are intended to be administered to subjects in an amount sufficient to be physiologically effective. An amount is referred to as physiologically effective if the dosage, route of administration, etc. of the composition are 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 said to be physiologically effective when administered in a dosage sufficient to suppress inflammation.

Further, the antibodies to ICAM-3 or fragments thereof can be administered either alone or in combination with one or more immunosuppressive agents (particularly recipients of transplanted organ or tissue). Administration of such a 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, shortly after or shortly after organ or tissue transplantation, but before any symptom of organ rejection). 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 a symptom of actual inflammation (such as rejection of an organ or tissue). Therapeutic administration of the compounds serves to attenuate any simultaneous inflammation (such as rejection of a transplanted whole organ or tissue, say, kidney, or non-jawed organ, say, 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. Suitable vehicles and their formulations, 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).

-27E 283478 B6 (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 is made to British Patent Applications Nos. 9009548.0, 9009549.8, and PCT Application Nos. PCT / US91 / 02942 and PCT / US91 / 02946, filed at the US Admissions Office on April 27, 1991.

Other pharmaceutical methods can be used to control the duration of action. Controlled release formulations may 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 appropriate macromolecules! by varying the concentration of embedded macromolecules and methods of their incorporation. Another possible method of controlling the controlled release time into the polymeric material particles, 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 enclosed in microcapsules prepared, for example, by coacervation or phase bound polymerization, in gelatin or poly (methyl methacrylate) capsules, or in colloidal drug delivery systems such as liposomes, albumin microspheres. microemulsions, nanoparticles and nanocapsules, or in macroemulsions.

Using the invention, a pharmaceutical composition comprising (a) an anti-inflammatory agent (such as an antibody capable of binding to ICAM-3, a fragment of said antibody capable of binding to ICAM-3, ICAM-3, a functional derivative of ICAM-3 or neimuno25 globulin) an ICAM-3 antagonist other than ICAM-I 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

Having described the invention in general terms, the compositions of the invention and methods for their preparation will be explained in more detail with reference to non-limiting examples.

Example 1

Characterization of ICAM-3, a novel adhesive ligand for LFA-1

Adhesion of cell lines and lymphocytes, Dustin et al., Cold Spring Harbor Symp. Quant. Biol. 54, 753-765 (1989)), to plates coated with LFA-1, were performed as previously described. LFA-1, purified from JY lysates, was absorbed into 96-well microtiter plates (Linbrotitertek) at 1100 sites / pm ² . The non-specific binding sites were blocked with 1% BSA and washed with 45 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 exhibited 91% CD21, while culturing the cells in the presence of 10µg / ml phytohemagglutinin (PHA) generated PHA blasts. Cells were labeled with fluoro50 chromium BCECF (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, after which 10 ⁵ cells were transferred to each well. Cell lines showed adhesion to solid IFA-1 for 1 h at 37 ° C and cells without adhesion were aspirated

6 times with needle # 23, while the lymphocytes were centrifuged for 5 min at 30 x g 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 Ψ between each flush. Flushing was more effective in perfectly 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 saturation concentration (1/200 dilution of ascites) 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) et al., J. Exp. Med. (1991) (in press)) and CBR-IC3 / l (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 SKW3 (Gefter et al., Som. Cell Gen. 3, 231-236 (1977)) and 600 hybridomas were screened for ability to inhibit SKW3 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 were recovered from established cells and contaminating erythrocytes were removed by hypotonic decomposition. Lymphocytes and monocytes were separated by cytometric analysis using direct and perpendicular light scattering, confirming specific MAb monocytes 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 with an EPICS V flow cytometer and fluorescence determined using EPICS Immune-Brite fluorescent beads (Coulter) to calibrate the cytometer. The expression of ICAM-3 on resting lymphocytes was 2 to 3 times greater than CD3 or LFA-1, while monocytes expressed 3 to 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.

-29GB 283478 B6

Table 2

Relative surface antigen expression of ICAM by immunofluorescence flow cytometry specific linear fluorescence intensity *

cell line / /type aICAM-1 (RR1 / 1) aICAM-2 (CBR-IC1 / 1) aICAM-3 (CBR-IC3 / 2) resting lymphocytes 4 22nd 106 PHA day blasts 22nd 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 Jurkat 2 166 161 Sup T 10 113 19 Dec Molt 4 1 242 117 lymphoblastoid B JY 154 119 47 SLA 224 113 306 Ramos 132 136 112 Paradise 266 88 0 monocytic U937 62 67 37 HL60 17 43 203 we had yes BK 896 3 0 RPMI 7591 475 0 0 erythroleukemic K562 293 143 0 different* ** 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 cytometer and one unit is approximately 10 ³ fluorescein equivalents (Coulte Diagnostics, Hialeah, FL).

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

-30GB 283478 B6

Example 3

Immunoprecipitation of ICAM-3

Cell surface staining with ^I25 I was performed with iodogen (Kishimoto et al., J. Biol. Chem. 264, 3588-3595 (1989)). Cells were lysed with tritone X-100 (1%). Lysates were pre-pretreated with Sepharose with bound bovine IgG 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 for acrylic lamellar gel poles on a 7% vertical slope plate (Laemmli, UK, Nature 227, 680685 (1970)) and proteins were visualized by autoradiography. Treatment of samples 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 of the peptide chain. Class 1 MHC contains N-linked carbohydrate, while ICAM-2 (mr 60000, 6 N-linked sites, chain M _r 28383) is highly glycosylated.

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. Table 3 summarizes the cell types that exhibit surface expression of ICAM-3. Expression of ICAM-3 appears to be limited to hematopoietic cells. Flow cytometry data was 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 the expression of ICAM-3 appears to be limited to hemopoietic cells.

-31 GB 283478 B6

Table 3

Relative surface antigen expression of ICAM by immunofluorescence flow cytometry specific linear fluorescence intensity *

cell line / /type aICAM-1 (RR1 / 1) aICAM-2 (CBR-IC1 / 1) aICAM-3 (CBR-IC3 / 2) resting lymphocytes 4 22nd 106 1 day PHA blasts 22nd 18 78 3-day PHA blasts 85 42 205 5-day PHA blasts 25 45 223 monocytes 13 39 224 neutroflly 1 0 213 lymphoblastoid T SKW3 0 98 273 Jurkat 2 166 161 SupT 10 113 19 Dec Molt4 1 242 117 lymphoblastoid B JY 154 119 47 SLA 224 113 308 Ramos 132 136 112 Paradise 266 88 0 monocytic U937 62 67 37 HL60 17 43 203 melanomas BK 896 3 0 RPMI 7591 475 0 0 erythroleukemic K562 293 143 0 different* ** 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 ³ fluorescein equivalents (Coulte Diagnostics, Hialeah, FL).

** Various cell lines include: human umbilical vein endothelial cells (hevec), 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.

- 32 GB 283478 B6

Example 5

ICAM-3 purification

ICAM-3 was purified by immunoaffinity chromatography, where the antibody to ICAM-3 CBRIC3 / 1 was immobilized by known methods on a matrix. Various sources were used as starting material for ICAM-3 isolation. These include, but are not limited to, SKW3, a human thymoma cell line, and a human tonsil. For the purification of ICAM-3, essentially the same methods as deFougerolles et al., J. Exp. Copper. 175: 185-190 (1992). The elution wash regimen is readily determined by one skilled in the art (the reagent varies slightly with each purified antigen and with each antibody used in immunoaffinity chromatography).

As further noted, ICAM-3, purified from SKW3 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 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 ICAM-3 molecular weight on various cell types. The molecular weight of ICAM-3 immunoprecipitated from neutrophils and immunoprecipitated from lymphocytes was found to differ. 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 to 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. Thus, changes in the molecular weight of ICAM-3 are highly likely due to differences in glycosylation. This can be readily confirmed by one skilled in the art by subjecting ICAM-3, isolated from neutrophils, to various glycosidases and other enzymes, and observing an 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 protein, which was purified from SKW3 or almond cells as above. Monoclonal antibodies were generated from immunized animals in a known manner.

-33 GB 283478 B6

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 SK.W3 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 SK.W3 cells to immobilized purified ICAM-3.

One of the evidence that a third LFA-1 ligand exists was the presence of an ICAM1 / ICAM-22 independent pathway for PMA-stimulated SK.W3 cell aggregation. We were able to block this aggregation either by mouse polyclonal antiserum to ICAM-3 or by combination of CBR-IC3 / 1 and CBRIC3 / 2.

Table 4 mAb ICAM-3 induced PMA aggregation *

mAb isotype itself + ICl + 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 nestanov. 5 5 5 antiserum ICAM-3 3 0 0 CBR-IC3 / 1 IgG1, k 5 4 - HP2 / 19 ** 1

* Refers to the extent of aggregation in J. Exp. Copper. 19910 - no aggregation ** Other mAb ICAM-3 from 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). ICAM-2 antibody (CBR-IC2 / 2) and ICAM-3 antibody (combination of CBR-IC3 / 1 and CBR-IC3 / 2) were tested for the ability to block (1) PMAstimulated adhesion of SK.W3 cells 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 and thus increase its ability to bind to ICAM-1 (Dustin et al., Nature 341, 619 (1989)). Giant. 5 demonstrates that a similar activation mechanism is present in LFA-1 / ICAM-3 binding.

-34GB 283478 B6

The ability of various antibodies to block binding of PMA stimulated SKW3 cells to purified ICAM-1 and ICAM-3 was examined. CBR-IC3 / 1 and CBR-IC3 / 2, when present individually, do not effectively block the binding of PMA-stimulated SKW3 cells to purified ICAM-3. However, binding of PMAstimulated SKW3 cells to ICAM-3 can be blocked using a combination of the two antibodies. As reported by deFougerolles in J. Exp. Med., CBRIC / 3/1 monoclonal antibody itself was able to block ICAM-3 binding to purified LFA-1.

The binding of PMA-stimulated SKW3 cells to ICAM-3 was further analyzed to determine whether it is temperature and cation dependent. As with ICAM-1, ICAM3 / LFA-1 binding was found to be temperature dependent (see Figure 8) and cation dependent (data not shown).

Giant. 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 a combination of antibodies to ICAM-1 and antibodies to ICAM-2 had little effect on PHA-stimulated cell division. However, a combination of 1) ICAM-1, ICAM-2 and ICAM-3 antibodies, 2) two ICAM-3 antibodies (IC3 / 1 and IC3 / 2), and 3) ICAM-1 and ICAM-3 antibodies 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 the response level using the proliferation of the corresponding cells as an index and has been described previously (Krensky et al., J. Immunol. 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 Figure 10. In summary, the antibody to ICAM-3 alone showed a low level effect. However, a higher level of MLR blocking efficacy has been found to be associated 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 tonsil cells as shown in Example 5. Purified ICAM-3 was enzymatically digested with Lys-C by known methods and the peptide fragments were resolved by HPLC. Giant. 11 is a gas chromatogram of various protein peaks observed during typical digestion. Peaks 10 and 17 were identified as containing peptide fragments of sufficient size and structure to allow sequencing (hereinafter referred to as the N-10 and N-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 to be KIDRATCPQHLK (SEQ ID NO: 1) for the presence of the first lysine (K) residue shown in the sequence, and Lys-C was thought to cleave proteins downstream of the lysine residues.

-35 GB 283478 B6

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.

Comparison 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 has 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.

io Example 11

ICAM-3 cDNA cloning

Based on the above peptide sequences, degenerate oligo15 nucleotide 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 nucleotide sequences of the probe based on the amino acid sequences of the NK-17 and NK10 proteins are:

G

G A A T

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

ProbeNK-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 as described previously (Wong 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.e. clone 11.2, a 35 insert region of about 1.6 to 1.8 kb was found. A graphical representation of clone 11.2 and the location of the NK-10 and NK-17 peptides is shown in Figure 12.

The ends of this insert were sequenced by known methods using primers derived from the flanking vector, while a 40 NK-10 probe was used as 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 Figure 18. First, a sequence with a high degree of homology of 45 to the transmembrane region of ICAM-1 has been identified. If production of soluble fragments is desired

Thus, all sequences beyond the beginning of the transmembrane region shown in Figure 12 should be deleted. 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 shown by sequence homologies that exist between ICAM-1, ICAM-2, and ICAM-3.

Example 12

Sequence homology between different ICAM molecules

-36GB 283478 B6

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

The results show that ICAM-3 shows a high level of homology with both ICAM-1 and ICAM2. Sequence homology between ICAM-1 and ICAM-3 was found in the first (Fig. 16) and in the fourth / fifth region of ICAM-1 as well as in the transmembrane region and partially cytoplasmic region of ICAM-1 (Figures 13 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).

Gene 11, 2 were used to further screen the libraries. Additional clones were found containing the inserted cDNA regions of about 2 to 2.4 kD. In all likelihood, clones are full-length cDNAs since no longer features have been identified.

List of sequences (1) General 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) mailing address:

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

(C) City: Washington (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: PatentIn Release # 1.0, version # 1.25 (vi) current application data :

(A) Application Number: US 92/04896 / PCT (B) Filing Date: 11.06.92 (C) Classification: A 61 K 39/00 (vii) Previous Application Date:

(A) Application Number: US 07 / 712,879 (B) Filing Date: 06.06.91

-37EN 283478 B6 (viii) 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:

(1) 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. SEQ ID NO. 1:

G GAG TTC CTG TTG CGG GTG GTC CCC CAG AAC CCT

Glu Phe Leu Arg Val Glu Pro Gin Asn Ser Val 15 1015

GGA GGG TCC CTG TTT GTG

Gly Gly Ser Leu Phe Val Cn Ser Ser Thr Asp Cys Ser Ser 25 2530

GAG AAA ATC GCC TTG

Glu Lys Ilu Ala Leu Glu Leu Ser Lys Glu Leu Val Ala 35 4045

AGG GGC ATG GGC TGG G152

Ser Gly Met Gly Trp (2) Information for sequence number 2:

(i) sequence characterization:

(A) length: 50 amino acids (B) type: amino acid (D) topology: linear

(Ii) molecule type: protein (xi) sequence description: id. SEQ ID NO. 2:

Glu Phe Leu Arg Val Glu Pro Gin Asn Ser Val

5 1015

Gly Gly Ser Leu Phe Val Cn Ser Ser Thr Asp Cys Pro Ser Ser

2530

Glu Lys Ile Ala Leu

4045

Ser Gly Met Gly Trp (2) Information for Sequence ID 3:

(i) sequence characterization:

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

(A) name / key: CDS (B) location: 2.189 (xi) sequence description: id. SEQ ID NO. 3:

C CCA TTG AGG GGT TCC ACA GTG ACC GTG AGG TGG ATG GCT GGG GCT Pro Leu Arg Gly Ser Thr Val Thr Val Ser Cys Met Ala Gly Ala 15 1015

CG GTC CG GTC ACG CTG GAC GGA GTT

Arg Gin Val Thr Leu Asp Gly Val Pro Ala Ala Ala Pro Gly 20 2530

CAG CCA GCT GAC GAC GAC

Gin Pro Ala Gin Leu Asin Ala Thr Glu Ser Asp AspGly

4045

CGC AGC TTC TTC TGC AGT

Arg Ser Phe Phe Cys. Ser Ala Thr Leu Glu Val His Gly GinPhe

5560

TTG CAG AG189

Leu Gin

-39GB 283478 B6 (2) Information for sequence number 4:

(1) sequence characterization:

(A) length: 62 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (xi) sequence description: id. SEQ ID NO. 4:

Pro Leu Arg Gly Ser

5 1015

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

2530

Gin Pro Ala Gin Leu Asin Ala Thr Glu Ser Asp Asp Gly

4045

Arg Ser Phe Phe. Cys Ser Ala Thr. Leu Glu Val His Gly Gin Phe

5560

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) characteristic:

(A) name / key: CDS (B) location: 1..113 (xi) sequence description: id. SEQ ID NO. 5:

ACT TTG TCG CCG GTC TTC GTG

Thr Leu Ser Phu Val Ala Val Leu Le Thr Leu Gly Val

1015

GTG ACT ATC GTA CTG GCC TTA

Val Thr Ile Val Thu Ala Leu Met Thr Val Phe Arg Glu His Gin

2530

CGG AGC GGC AGT

Arg Gly Ser Tyr His Val

-40GB 283478 B6 (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. SEQ ID NO. 6:

Thr Leu Ser Pro Val Phe Val Thu Leu Thr Leu Gly Val 15 10 15

Val Thr Ile Val Leu Ala Leu Met Tyr Val Phe Glu His Gin 20 25 30

Arg Gly Ser Tyr His Val

-41 GB 283478 B6

Claims (25)

PATENT CLAIMS A recombinant DNA molecule capable of encoding ICAM-3 or a functional derivative thereof, wherein said DNA molecule comprises at least one fragment selected from the group consisting of the following nucleotide sequences: GG TTC CTT TTG CGG GTG GAG CCC CAG AAC CCT Glu Phe Leu Argu Glu Pro Gin Asn Ser GGA GGG TCC CTG TTT GTG AAC Gly Gly Ser Leu Phe Val Asn Cys Ser Thr Asp Cys For Ser Ser Glu 20 2530 AAA ATC GCC TTG GG ACG TCC CTA TCA AAG GAG Lys Ile Ala Le Glu Thr Ser Leu Ser Lys Glu Leu Val Ala Ser Gly 35 4045 ATGGGCTGGG152 Met Gly Trp C CCA TTG AGG GGT TCC ACA GTG Pro Leu Arg Gly Ser Thr Val Ser Cys Met Ala Gly Ala 15 1015 CAG GTC CAG GTC ACG CTG GAC GGA GTT CCG GCC GCG GCC CCG GGG CAG 94 Arg Val Gin Val Thr Leu Asp Gly Val Pro Ala Ala Ala Pro Gly Gin 20 2530 CCA GCT CAA CTT CAG CTA AAT GCT ACC GAG GAC GAC GGA GC CGC AGC 142 Pro Ala Gin Leu Gin Leu Asn Ala Thr Glu Ser Asp Asp Gly Arg Ser 35 4045 TTC TTC TGC AGG GTC ACT CTC GAG GTG CAC Phe Phe Cys Ser 50 5560 ACT TTG TCC CCG GTC TTC GTG GCG GTG TTA CTG ACC Thr Leu Ser Phu Val Ala Val Leu Le Thr Leu Gly Val Val 15 1015 ACT ATC GTA CTG GCC TTA ATG TAC GTC TTC AGG GAG CAC CAA CGG AGC 96 Thr Ile Val Leu 20 May 25 30 GGC AGT 113 Gly Ser Tyr His Val 35 -42 GB 283478 B6 A functional derivative of ICAM-3, wherein said derivative is an ICAM-3 fragment selected from the group consisting of the following amino acid sequences: Glu Phe Leu Leu Arg Val Glu For Gin Asn For Val Leu Ser Ala Gly 1 5 1015 Gly Ser Leu Phe Val Asn Cys Ser Thr Asp Cys For Ser Ser Glu Lys 20 2530 Ile Ala Le Glu Thr Ser Leu Ser Lys Glu Leu Val Ala Ser Gly Met 35 4045 Gly Trp, Pro Leu Arg Gly Ser Thr Val Ser Cys Met Ala Gly Ala Arg 15 1015 Gly Gin Pro Gly Gin Pro Gly Gin Pro 20 2530 Ala Gin Leu Gin Leu Asn Ala Thr Glu Ser Asp Asp Phly 35 4045 Phe Cys Ser Ala Thr Le Glu Glu Val His Gly Phe Le Gin 50 5560 Thr Leu Ser Phu Val Val Val Leu Le Thr Leu Gly Val Val 15 1015 Thr Ile Val Leu Met Le G Met His Gin Arg Ser 20 2530 Gly Ser Tyr His Val. The recombinant DNA molecule of claim 1, further capable of expressing in the cell ICAM-3 or a functional derivative thereof. The functional derivative of claim 2, wherein said fragment is selected from the group consisting of D1, D1-2, DI-3, D1-4 and D1-5 ICAM-3. ICAM-3 or a functional derivative thereof, obtained by expressing the recombinant DNA molecule of claim 3 in a cell. A method for producing ICAM-3 or a functional derivative thereof, characterized in that the recombinant DNA molecule of claim 3 is expressed in a cell. 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 7, wherein binding of said antibody to said molecule reduces the ability of said molecule to bind to a receptor molecule. The antibody of claim 8, wherein the receptor is LFA-1. The antibody of claim 8, wherein the receptor is MAC-1. -43 GB 283478 B6 The antibody of claim 8, wherein the receptor is p150.95. A method of producing a desired hybridoma cell that produces an antibody capable 5 bind to ICAM-3 or a functional derivative thereof, comprising the steps of a) immunizing the animal with purified ICAM-3; b) fusing the spleen cells of the animal with a myeloma cell line; (c) allowing fused 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. 13. A method for detecting the presence of a cell expressing ICAM-3, wherein: 20 se (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 with a complementary nucleic acid molecule present in said cell or in an extract of said cell, which method is performed in vitro. 30 14. A method of diagnosing the presence of ICAM-3 in a fluid sample taken from a mammalian subject, comprising: (a) incubating said biological fluid sample of said subject with a composition comprising an antibody or fragment thereof capable of binding to a cell expressing ICAM-3; and b) detecting said antibody bound to said fluid, the method being carried out in vitro. 40 15. The method of claim 15, wherein said fluid is selected from the group consisting of blood, serum, plasma, synovial fluid, amniotic fluid, spinal fluid, or urine. 16. A method for identifying agents capable of modulating the binding of ICAM-3 to LFA-1; 45, characterized in that ICAM-3 is contacted with LFA-1 in the presence of such a composition, and said modulation of ICAM-3 / LFA-1 binding is measured. 17. The method of claim 17 wherein LFA-1 and ICAM-3 are purified to remove naturally occurring impurities. The method of claim 17, wherein the LFA-1 is expressed on the cell surface and ICAM-3 is purified to remove naturally occurring impurities. -44 GB 283478 B6 The method of claim 17, wherein ICAM-3 is expressed on the cell surface and LFA-1 is purified to remove naturally occurring impurities. 20. A pharmaceutical composition for modulating cell biological functions mediated by ICAM-3, comprising a composition selected from the group consisting of an antibody capable of binding to ICAM-3, a fragment of such an 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. 21. The pharmaceutical composition of claim 21, further comprising an immunosuppressive agent. 22. A pharmaceutical composition comprising an ICAM-3 modulating agent in combination with a pharmaceutically acceptable carrier. 23. The pharmaceutical composition of claim 23, wherein the composition is in combination with at least one other immunosuppressive agent. 24. A therapeutic composition comprising the antibody molecule or fragment thereof of claim 7 in combination with a pharmaceutically acceptable diluent, vehicle or carrier. 25. A diagnostic composition comprising the antibody molecule or fragment thereof of claim 7 in a detectably labeled form.