BG61682B1 - Intercellular adhesion molecule-3 and its binding ligands - Google Patents

Abstract

The intercellular adhesion molecule-3 (ICAM-3) is includedin the process when the leukocytes identify the places ofinflammation and migrate to them in order to stick to the cellularsubstrates during the inflammation. The invention is related tothose molecules, to screening tests for their identification andto antibodies capable of binding them, as well as to therapeuticaland diagnostic applications of the adhesion molecules and theantibodies capable of binding them.

Description

BACKGROUND OF THE INVENTION

This patent is a partial extension of US No. 07/712 879 / filed June 11, 91 / and relates to US No. 07/045963 / filed 04.05.87 /, 07/115798 / filed 02.11.87 /, 07 / 155943 / filed 16.02.88 /, 07/189815 / filed 08.05.88 / and 07/250446 / filed 28.09.88 /, 07/454294 / filed 22.12.89 / and PCT Application No. PCT / US 91 / 02942 and PCT / US 91/02946 / filed on 04/27/91 /

FIELD OF THE INVENTION

The present invention relates to the discovery of a new intercellular adhesion molecule, designated ICAM-3, which is involved in the process in which a population of leukocytes recognizes and adheres to cellular substrates. ICAM-3 mediates cellular interactions with other lymphocytes, macrophages, and neutrophils at sites of inflammation and sites of immune responses.

The invention also relates to the use of ICAM-3 alone or in combination with ICAM-1 and / or ICAM-2 for inhibiting intercellular adhesion of cells of granulocyte, lymphocyte or macrophage origin. The use of such molecules provides a method of treating specific and non-specific inflammations.

The invention also relates to therapeutic and prophylactic methods of suppressing H1V lymphocyte infection, particularly HIV-1 in an individual who is HIV-infected or infected, by administering ICAM-3 alone or in combination with ICAM- 1 and / or ICAM-2. This provides therapy for diseases such as AIDS caused by the HIV virus.

The invention also relates to a therapeutic method for inhibiting the migration of HIV-1 infected blood cells by ICAM-3 alone or in combination with ICAM-1 and / or ICAM-2. In this way, therapy is provided for diseases such as AIDS caused by the HIV-1 virus.

The invention also relates to a therapeutic method for suppressing cell death and formation of "syncytium" in an HIV infected individual by ICAM-3 alone or in combination with ICAM-1 and / or ICAM-2. Thus, therapy is provided for diseases such as AIDS caused by the HIV-1 virus.

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

In addition, the invention relates to molecules capable of binding to ICAM-3 (hereinafter referred to as anti-ICAM-3). The binding of an anti-ICAM-3 molecule to ICAM-3 is expected to modulate the biological functions associated with ICAM-3. The binding molecules of the invention may be an antibody, peptide or carbohydrate capable of binding to ICAM-3. Such binding molecules can be used to modulate the biological functions of ICAM-3.

The present invention also relates to the use of anti-ICAM-3, alone or in combination with anti-ICAM-1 and / or anti-ICAM-2, for inhibiting intercellular / intercellular / adhesion of cells of granulocyte, lymphocyte or macrophage origin . The use of such molecules provides a method for the treatment of specific and non-specific inflammations.

The invention also relates to therapeutic and prophylactic methods of suppressing the leukocyte infection of HIV, especially H1V-1, in individuals who are available for HIV or infected with H1V by administering anti-1CAM-3 alone or in combination with anti-ICAM-1 and / or anti-ICAM-2. This provides treatment for diseases such as AIDS caused by the HIV virus.

The invention further relates to a therapeutic method for inhibiting the migration of HIV-1 infected cells from the bloodstream by using an anti-ICAM-3 agent, alone or in combination with an anti-ICAM-1 and / or anti-ICAM-2 agent, so treatment is provided for diseases such as AIDS caused by the HIV virus.

The present invention also relates to the use of an anti-ICAM-3 agent, alone or in combination with anti-ICAM1 and / or anti-ICAM-2, for the treatment of asthma.

Description of the fields of the invention

A. Leukocyte adhesion and function

Leukocytes should be able to adhere to cellular substrates to protect the host well from foreign invasion, such as by bacteria or viruses, see p. Eisen, H.W., / In: Microbiology, 3rd Ed., Harper & Row, Philadelfia, PA / 1980 /, pp. 290-295 and 381-418 for an overview of these features. Leukocytes must be capable of adhering to endothelial cells so that they can migrate from the circulatory system to sites of inflammation. Furthermore, they must adhere to the antigen presenting cells so that a normal specific immune response can occur, and finally, they must adhere to a suitable cellular target so that lysis of the virus-infected cell occurs or the corresponding tumor cell.

The leukocyte surface modules involved in mediating the above related adhesion functions have been identified using the hybridoma technique. Briefly, human T-cell monoclonal antibodies / Davignon, D. et al., Proc. Natl.Acad.Sci, USA 78: 4535-4539 (1981) and murine splenocytes / Springer, T.et al. Eur.J.Immunol. 9: 301-306 (1979) have been identified as being found to bind to the leukocyte surface and to inhibit adhesion-related functions described above / Springer, T. et al., Fed.Proc. 44: 2660-2663 / 1985 //. The molecules identified by such antibodies are called Mass-1 and lymphocyte-functionally associated antigen. 1 / 1FA-1 /. Mass-1 is a heterodimer found in macrophages, granulocytes and large granular lymphocytes. IFA-1 is a heterodimer found in most lymphocytes / Springer, T.A. et al. Immunol.Rev. 68: 111-135 / 1982 //. These two molecules, plus a third molecule, p150.95, which has tissue distribution similar to Mass-1 /, play a role in cellular adhesion (Keizer, G. et al., Eur.J.Immunol. 15: 11421147/1985 //.

The leukocyte molecules described have been found to be members of the glycoprotein family / Sanches-Madrid, F.et al., J.Exper.Med. 158: 1785-1803 (1983); Keizer, G.D et al., Eur.J.Immol. 15: 1142-1147 (1985), designated "CD-28 family" of glycoproteins. This glycoprotein family is composed of heterodimers having one α and one β-chain. Although the α-chain of each of the antigens is different, the β-chain is very well conserved / SanchesMadrid, F. et al., J.Exp.Med. 158: 1785-1803 I 1983 //. The β-chain of the CD18 glycoprotein family has a molecular weight of 95 kd, while the α-chains range from 150 kd to 180 kd / Springer, T., Fed.Proc. 44: 2660-2663 / 1985 //. Although the α-subunits of the membrane proteins do not share the extensive homology shared by the β-subunits, analysis of the α-subunits of the glycoproteins shows that there is a substantial similarity between them. A review of the analogies between the α and β-subunits of IFA-1-linked glycoproteins is made by Sanches-Madrid, F. et al., / J.Exper.Med. 158: 586-602 (1983); J.Exper. Med. 158: 1785-1803 / 1983 //.

A group of individuals have been identified that are unable to express normal amounts of any member of this family of adhesion proteins on the cell surface of their leukocytes / Anderson, D.C. et al., Fed.Proc. 44: 2671-2677 (1985); Anderson, D.C. et al., J.Infect.Dis. 152: 668-669 / 1985 //. Such individuals are said to be suffering from leukocyte adherence deficiency disease (LAD) /Anderson, D.C., et al., Fed. Proc. 44: 2671-2677 (1985); Anderson, D.C., et al., J.Infect.Dis. 152: 668-689 (1985). Characteristic features of patients suffering from LAD include necrotic soft tissue damage, pus formation and poor wound healing, as well as abnormality of adhesion-dependent leukocyte functions in vitro, and susceptibility to chronic and recurrent bacterial infections. The granulocytes from these LAD patients behave in the same defective manner in vitro as their normal counterparts do in the anti-CD18 monoclonal antibody. Ie they are unable to represent adhesion-related functions such as aggregation or adhesion to endothelial cells. More important, however, is the observation that these patients are unable to elicit a normal inflammatory response due to the inability of their granulocytes to attach to cellular substrates. Most important is the observation that granulocytes from these LAD patients are unable to reach the sites of inflammation, skin infections, for example, due to their inability to attach to endothelial cells in blood vessels near inflammatory lesions. Such attachment is a necessary step for extravasation.

The lymphocytes of these patients show in vitro defects, similar to their normal relatives, whose CD-18 family of molecules is antagonized by antibodies. Moreover, these individuals are unable to attain a normal immune response due to their cells' inability to adhere to cellular substrates / Anderson, D.C. et al., Fed.Proc. 44: 2671-2677 (1985); Anderson, D.C., Etal., J.Infect.Dis. 152: 668-689 / 1985 //. These data indicate that immune responses are upset when lymphocytes are unable to adhere normally due to the absence of functional adhesion molecules of the CD-18 family.

In general, the ability of leukocytes to maintain the health and vitality of animals requires that they be able to adhere to other cells (such as endothelial cells). Such adherence has been found to require cell-cell contact, which involves specific receptor molecules on the surface of leukocytes. These receptors allow leukocytes to adhere to other leukocytes, to endothelial cells, and to other non-vascular cells. The cell surface receptor molecules, LFA1, Mass-1 and pl50,95, are strongly bound to each other. People whose leukocytes do not have these surface molecular receptors exhibit chronic and recurrent infections, as well as other clinical symptoms, including defective antibody responses.

In addition, insofar as leukocyte adhesion is involved in the process in which foreign tissue is identified and rejected, understanding of this process is applicable in the field of organ transplantation, such as kidney, bone marrow, tissue grafting, in allergy and oncology.

B. HIV infection

HIV infection is the cause of AIDS. Many variants of HIV have been described, with the big two being HIV-1 and HIV-2. HIV-1 is prevalent in North America and Europe and HIV-2 is found only in Africa. Viruses have similar structures and encode proteins with similar functions. HIV infection is thought to occur by binding the viral protein (labeled gpl 20) to the receptor molecule (labeled CD4) present on the surface of T4 (T-helper / lymphocytes) Schnittmann, SMet al., J. Immunol . 141: 41814186/1988 /. After binding to this receptor, the virus enters the cell and replicates, killing the T cell during this process. Destruction of the individual's T4 population is a direct result of HIV infection.

T-cell destruction leads to a worsening of the ability of infected patients to respond to accidental infections. Although individuals affected by AIDS often develop cancer, the link between this cancer and HIV infection is in most cases doubtful.

Although the actual replication of the HIV virus is lethal to infected cells, such replication is only found in a small fraction of the T4 cells of an infected individual. Recent results suggest more viremia than previously reported, and the incidence of T-cell infections is about 1%.

Several lines of research have elucidated other mechanisms by which the HIV virus mediates the destruction of the T4 population.

Apart from HIV replication, HIV-infected cells can be destroyed by cytotoxic killing cells. These killer cells are normally present in humans and serve to register the functions of the host, destroying any foreign cells (such as blood transfusions or organ transplants) that have not been previously reported.

In HIV infection, T4 cells show a gpl 20 molecule on their cell surface. Killer cells recognize such T4 cells as foreign (relative to their own body) and accordingly mediate their destruction.

HIV infection can also lead to the destruction of uninfected healthy cells. Infected cells can secrete gpl 20 protein in the bloodstream. The free gpl 20 molecules can then bind to the CD4 receptors of healthy, uninfected cells. Such binding causes the cells to catch the appearance of HIV-infected cells. Cytotoxic, killer cells recognize gpl20 binding to uninfected T4 cells, concluding that these are foreign to the body and mediate their destruction.

Another mechanism of particular relevance to the present invention in which HIV can cause T4 cell death is through the formation of syncytium. Syncytium is a multinuclear giant cell. formed by the fusion of more than a few hundred T4 cells. H1V infection causes this cell to fuse with other T4 cells, both infected with H1V and healthy ones. The syncytium cannot function and soon dies. His death causes destruction of both HIV-infected and uninfected cells. This process is of particular importance to the present invention insofar as it results in direct cell-cell contact. The ability of HIV-infected cells to form syncytium indicates that such cells acquire a way to fuse with healthy cells.

HIV infection, in particular HIV-1, apparently affects the lymphocyte cell surface responses, in particular the pooling and fusion reactions mediated by these heterodimers / Petit, A. J., et al., J. Clin, Invest. 79: 188 (1987); Hildreth, J. E. K. et al., Science 244: 1075 (1989); Valentin, A., et al., J. Immunology 144: 934-937 (1990); Rossen, R.D., et al., Trans. Assoc. American Physician 102: 117130/1989 /. Following HIV-1 infection, homotypic aggregation of U937 cells increased, as did expression of CD18 and CD11b on the cell surface / Petit, A.J., et al. J. Clin.Invest 79: 188/1987 //. HIV-1 infected U-937 cells adhere to U-1, stimulate the endothelium at a higher frequency than uninfected U-937 cells; this behavior can be suppressed by treating infected cells with anti-CD18 or anti-CD11a monoclonal antibodies or by treating endothelial substrates with anti-CDAM-1 antibodies / Rossen, RD, et al., Tran.Assoc.American Phisician 102: 117-130 / 1989 //. Monoclonal antibodies against CD 18 or CD11a have also been found to be capable of inhibiting syncytium formation, including phytohaemagglutins (PHA) -stimulated lymphoblastic cells and constitutively infected, CD-4-negative T cells / Hildredth, JEK, et al., Science. 244: 1075/1989 //. Treatment of only virus-infected cells with anti-CD18 or anti-CD11a monoclonal antibodies has been found to have only a small effect on syncytium formation, suggesting that these antibodies generally protect uninfected target cells from infection / Hildreth, JEK, et al., Science 244: 1075 (1989); Valentin, A., et al., J. Immunology 144: 934-937 / 1990 //. Valentin et al. these observations have recently been confirmed by demonstrating the CD18-specific monoclonal antibody inhibition of syncytium formed by re-cultivation of persistent T-cell lines with HIV-1 infected U937 cells.

Although the mechanism by which monoclonal antibodies specific for CD 18 or CD 11 protect susceptible cells from fusion with HIV-infected cells remains unknown and is not necessary within the meaning of the present invention, radiological studies of labeled gpl20 suggest that heterodimers containing CD 18 do not provide a binding site for the virus (Valentin, A., et al., J. Immunology 144: 934937 (1990). That's right. HIV infection involves cell-cell interactions, and / or virus-cell interactions that mimic cell-cell interactions. These cell-cell interactions can lead to the transport of virus-free cells or of virus-infected cells through the endothelial barriers. Viral cell interactions that mimic cellular cells may facilitate or enable the free virus to attach to / or infect healthy cells.

The present invention thus proceeds in part from the observation that HIV infection, in particular HIV-1 infection, results in increased expression of the CD11a / CD18 heterodimers and their binding ligands. This increased expression is of particular importance because it enhances the ability of HIV-infected T cells to adhere to or aggregate with each other / i.e. to undergo homotypic aggregation. Insofar as such homotypic aggregation was not observed among normal leukocytes, this finding indicates that expression of the CD11 / CD18 receptors and / or ligands, such as ICAM-1, is required for such aggregation. IFA-1 must bind to 1CAM-1 in order to exhibit homotypic aggregation. As shown here, ICAM-3 is the only member of the ICAM family of molecules that are highly expressed in dormant T cells (resting T cells). Only anti-ICAM-3 antibodies are capable of blocking the attachment of T cells to IFA-1 if the T cells are not "activated". Therefore, anti-ICAM3 antibodies can be used to inhibit T-cell aggregation.

In addition, anti-1CAM-3 antibodies can be used to block the adhesion processes of infected T cells, which allows HIV-1 to be able to be transferred from an infected cell to healthy cells of the individual and also allows or facilitates infection of healthy cells with free virus.

C. Migration of HIV infected cells

The migration and spread of leukocytes are important in protecting the individual from the effects of the infection. However, these processes are responsible for the migration and spread of virus-infected leukocytes. Of particular interest is the migration and spread of HIV-infected leukocytes. Migration of such cells leads to the formation of extravascular foci and may cause tumors and other neoplasms.

Histological studies have shown that the affected organs have extravascular mononuclear cell infiltrates. Attempts to identify such virus-infected cells in similar infiltrates in the central nervous system have shown the presence of HIV-infected cells. These studies have shown that H1V-1 viruses exist primarily in monocytes and macrophages, as well as in other cells of this type (R.T.Jonnson, et al., FASEB I. 2: 2970 (1988); M.H.Stoler et al., J.Amer.Med.Assn. 256-2360 / 1986 / S.Gartner et al. Science 233: 215/1986 //.

The mechanisms that stimulate the formation of extravascular infiltrates of HIV-1-infected monocytoid cells have not been well defined previously. Mechanisms may include both the transport of virus-free cells and the transport of the virus through endothelial barriers within the cytoplasm of infected mononuclear cells.

Inasmuch as infection with HIV-1 stimulates cell surface expression of molecules, facilitating attachment of leukocytes to vascular endothelial cells and translocation of leukocytes from blood to extravascular tissue sites / C. W. Smith et al., J. Clin. Invest. 82: 1746 (1988), incorporated herein by reference, it has been proposed to use antibodies that inhibit cell migration in order to prevent the spread of HIV-infected cells (WO 90/13316).

D. Asthma. Clinical characteristics.

Asthma is a heterogeneous family of diseases. It is characterized by hyperreactivity of the tracheobronchial stimuli. / Cow, A.V., Allroy and Inflammation, Academic Press NY / 1987 /. Clinically, asthma is manifested by extensive narrowing of the tracheobronchus, sticky thick secretions, cramps, coughs and suffocation. Although the contributing causes are unknown, the end result is an increase in airway resistance, hyperinflammation of the tonsils and thorax, and incorrect distribution of ventilation and pulmonary blood flow. The disease manifests itself in episodic periods of acute symptoms, distributed between periods free of symptoms. Sharp episodes lead to hypoxia and can be fatal. Approximately 3% of the world's total human population suffers from asthma.

Two types of asthma have been described: allergic asthma and idiosyncratic asthma. Allergic asthma is usually associated with congenital allergic diseases such as rhinitis, tracheitis, urticaria, eczema and more. The condition is characterized by a positive reaction (inflammation of the skin) upon intradermal injection of airborne antigens such as pollen, environmental or occupational pollutants, etc., and increased levels of serum IgE. The development of allergic asthma appears to be causally associated with the presence of IgE antibodies in many patients. Patients with asthma who do not show the characteristic reactions described above are considered to suffer from idiosyncratic asthma.

Allergic asthma is thought to be dependent on the IgE response, controlled by T- and Vlimfocytes and activated by the interaction of airborne antigens with the germ of pre-formed IgE molecules. An antigenic collision can occur at concentrations sufficient to produce IgE for a prolonged period of time to make the individual sensitive. Once sensitive, an asthma patient may show symptoms in response to extremely low levels of the antigen.

Asthma symptoms can be exacerbated by the presence of key antigens, environmental factors, occupational factors, physical exertion, or emotional stress.

Asthma can be treated with methylxanthines (such as theophylline, β-adrenergic competitors) such as catecholamines, resorcinol, saligens and ephedrine /, glucocorticoids (such as hydrocortisone), fatty cell degranulation inhibitors / such as chrominol, e.g. atropine.

Asthma is thought to involve an influx of eosinophils / '' eosinophilia '' into the tissues of the tonsils / Frigas, E. et al., J. Allergy Clyn. Immunol. 77: 526-537 (1986).

The understanding of the immunological basis of asthma is derived from the results of bronchoalveolar studies / Godard, P. et al., J. Allergy Clyn.Immunol. 70:88 / 1982 // and studies on respiratory smooth muscle tissue devoid of epithelium / Flavahan, N.A / et al., J.Appl.Physiol. 58: 834 (1985); Barnes, P.J. et al. Br.J.Pharmacol. 86: 685/1985 //. Although these studies do not elucidate the mechanism underlying the asthma immunology, they have led to the development of a basic accepted hypothesis regarding the immunological etiology of the disease / see. Frigas, E.et al., J. Allergy Clyn., Immunol. 77: 527-537 / 1986 //.

The pathology of asthma is closely related to the massive infiltration of the eosinophil pancreas parenchyma and its mucociliary destruction.

The "eosinophilic hypothesis" implies that eosinophils are attracted to the bronchi in order to neutralize the harmful mediators released by the fat cells of the tonsils. According to the hypothesis, eosinophils are attracted to the bronchi, where they degranulate to release cytotoxic molecules. In degranulation, eosinophils release enzymes, for example, histaminase, arisulfatase, and phospholipase D, which enzymatically neutralize harmful mediators of fat cells. These molecules also destroy the mucociliary apparatus, thus preventing the clearance of bronchial secretion and contributing to the characteristic damage of the tonsils to asthma.

Insofar as asthma involves the migration of cells, it has been suggested to use antibodies to inhibit this migration and to modify the effect of the allergens in the subject (WO 90/10453).

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a new cell adhesion molecule referred to as an intracellular adhesion molecule-3 (ICAM-3). In addition, the invention relates to functional derivatives of ICAM-3, anti-ICAM-3 antibodies, fragments of these antibodies, humanized anti-ICAM-3 antibodies and other molecules capable of binding and inhibiting ICAM-3 functions.

The invention further relates to diagnostic and therapeutic applications of all the molecules described above.

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

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

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

The invention further provides a hybridoma cell capable of producing the monoclonal antibody described above.

The invention includes a method of producing the desired hybridoma cell that produces an antibody capable of binding ICAM-3 or its functional derivatives, which comprises the steps of:

a / immunization of an animal with an immunogen selected from the group consisting of: substantially pure ICAM-3, a cell expressing ICAM-3, a cell membrane of a cell expressing ICAM-3, ICAM-3 attached to a carrier, a peptide fragment of ICAM-3, or peptide fragments of ICAM-3 bound to a carrier.

c / fusion of splenocytes isolated from the immunized animal with a myeloma cell line, enabling fused splenocytes and myeloma cells to form antibody secreting hybridoma cells, and d / screening of hybridoma cells for such a hybridoma cell capable of produces an anti-ICAM-3 antibody.

The invention further relates to a method for modulating ICAM-3, the mediated biological function of the cell, wherein it is provided to provide an object in need of treatment with an effective amount of ICAM-3 modulating agent selected from the group consisting of: an antibody capable of binding to ICAM-3; a fragment of this antibody, which fragment is capable of attaching to ICAM-3; a functional derivative of ICAM-3; ICAM-3; and a non-immunoglobulin antagonist of 1AM-3 other than ICAM-1, ICAM-2, or a member of the CD-18 family of molecules.

The invention further provides a method of treating specific inflammation in humans and in other mammals, which involves providing such an amount of anti-inflammatory agent sufficient to suppress inflammation. The anti-inflammatory agent is selected from the group consisting of: an antibody capable of binding to ICAM-3, a fragment of that antibody; this fragment is capable of binding to a non-immunoglobulin antagonist of ICAM-3, wherein the inflammation in question is the result of organ transplantation (solid organs such as kidney) or transplantation of non-solid organs such as bone marrow or tissue planting.

In addition, the invention provides a method of treating non-specific inflammation in humans as well as in other mammals, where this method involves providing a subject in need of treating such an amount of an anti-inflammatory agent sufficient to suppress inflammation, where this anti-inflammatory agent is selected from the group consisting of: an antibody capable of binding to ICAM-3; an antibody fragment; this fragment is capable of binding to ICAM-3; substantially pure ICAM-3; a functional derivative of ICAM-3; or non-immunoglobulin antagonist of ICAM-3.

The invention also includes a method of treating inflammation in which inflammation is the result of the following diseases: age-related respiratory syndrome; multiple organ damage syndrome secondary to septicemia; multiple organ damage syndrome secondary to septicemia, hemorrhage or trauma; reperfusion damage to the myocardial or other tissues; acute glomerulonephritis; reactive arthritis; dermatosis with acutely inflammatory components; acute purulent meningitis or other central nervous system lesions, such as stroke, fever, hemodialysis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte-related transfusion syndrome, and cytokine-induced toxicity.

The invention further includes a method for suppressing hematopoietic tumor cell metastases, the cell using a member of CD-18 (in particular LFA-1) for migration, where this method involves providing a patient in need of treatment with such an amount of agent sufficient to suppress metastases. wherein this agent is selected from the group consisting of: an antibody capable of binding to ICAM-3; toxin production of this antibody; a fragment of this antibody, which fragment is capable of binding to ICAM-3; the toxin produced by this fragment, essentially pure ICAM-3; produced toxin from this fragment; a functional derivative of ICAM-3; produced toxin from this derivative (functional) of ICAM-3; or a non-immunoglobulin antagonist of ICAM-3 other than a member of the CD-18 family of molecules.

The invention further includes a method of inhibiting the growth of ICAM-3-expressing tumor cells, wherein a patient in need of treatment is provided with an amount of agent sufficient to suppress growth, and according to which method the agent is selected from the group consisting of : an antibody capable of binding to ICAM-3; a toxin derivative of this antibody; a fragment of a given antibody, such fragment being capable of binding to ICAM-3; a toxin derivative of a given fragment; toxin derivative, member of the CD-18 family of molecules; or a toxin derivative of the functional derivative of a member of the CD-18 family of molecules.

The invention also relates to a method for detecting the presence of cells expressing 1AM-3, which includes:

(a) incubating a cell or cell extract in the presence of a nucleic acid molecule capable of hybridization with ICAM-3 MPNA; and b) determining whether the nucleic acid molecule has hybridized to a complementary nucleic acid molecule present in the same cell or in the same cell extract.

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

a) incubating the sample with an antibody or fragment thereof capable of binding to ICAM-3; and / or whether the antibody binds the sample.

The invention further provides a pharmaceutical composition comprising:

a / an agent selected from the group consisting of: an antibody capable of binding to ICAM-3; a fragment of this antibody, which fragment is capable of binding to ICAM-3; substantially pure-1CAM-3; a functional derivative of ICAM-3; or a non-immunoglobulin antagonist of ICAM-3 other than a member of the CD-18 family of immunosuppressive agents.

Short description of the figures

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

Adhesion of cell lines (A) and lymphocytes (B) to purified LFA-1 was counted for 1AM-1DSAM-2 and ICAM-3. Binding of BCECF-labeled cells to LFA-1- coated microtiter plates in the presence of blocking MA _c specific for LFA-1, ICAM-2 and ICAM-3. The control plates were coated without LFA-1.

Figure 2. Low cytometric analysis of cellular ICAM-1, ICAM-2 and ICAM-3 expression.

(A) Lymphoblastoid cell lines were labeled with saturated amount or MA _in RR1 / 1 / anti-ICAM-1, MA _in CB-1C2 / 1 / anti1CAM-2 /, MA _b CBR-IC3 / 1 / anti-1CAM-3 (or unbound MA control _in hb3 (thin line), and then followed by FITC anti-mouse immunoglobulin. (B) The remaining human lymphocytes and 3-day activated lymphocytes were analyzed for ICAM expression as indicated above.

Figure 3. Immunoprecipitation of ICAM-3.

/ A / ^{| 25} 1 - labeled lysates immunoprecipitated with either non-binding control X63 of _MAb CB-IC3 / 1 / anti-ICAM1 3 /.

(B) ¹²⁵ 1-labeled SKW3 cell lysates treated with (+ / or without) - (N-glycanase) and immunoprecipitated with a non-binding control MA _in X63, MA _in W6 / 32 / anti-HEA-A, B, C / , MA _in RR1 / 1 / anti-ICAM-1 /, MA _b CBR-IC2 / 1 / anti-ICAM2 /, or MA CB-IC3 / 1 / anti-1AM-3 /.

Figure 4. Immunoprecipitation of ICAM-3 from various sources

ICAM-3 was immunoprecipitated from ¹²⁵ l-labeled lymphocytes and neutrophil lysates using mAv-double sepharose. The immunoprecipitated ICAM-3 was redissolved by polyacrylamide gel electrophoresis. Other mAv are used to precipitate other cell surface molecules. Human sepharose (control line), CBR-IC2 (1 / ICAM-2), CBR-1C3 (1) (ICAM-3), TS2 (4) (LFA-1), LM2 (1) (MAC-1).

Figure 5. Effect of PMA on SKW3 binding to ICAM-1 and ICA.M-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 described previously (Dustin et al., Nature 341: 619 (1989); Marlin et al., Cell 51: 813-819 (1987). An imaginary experiment is shown and the wrong bars show a standard deviation (SD).

Figure 6. Antibody blocking PMA stimulated SKW3 cell adhesion.

Various antibodies have been tested for their ability to block PMA stimulated SKW3 cells for binding to purified ICAM-1 or ICAM-3 as described previously (Dustin et al., Nature 341: 619 (1989); Marlin et al., Cell 51: 813-819 / 1987 //. One representative experiment is shown in the figure, with the standard error bars denoting one SD.

Figure 7. Binding of COS cell transfectants to purified ICAMs.

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

Figure 8. Temperature dependence of PMA stimulated SKW3 binding to ICAM.

The ability of PMA-stimulated SKW3 cells to bind to purified ICAM-1 and ICAM-3 was tested at 4,16,22 and 37 ° C as described previously. / Marlin et al., Cell 51; 813-819 / 1987 //. Tests were performed in the presence or absence of LFA-1 mAv (TS1 / 22). One representative experiment is shown, and the lines representing the standard error indicate SD.

Figure 9. Antibody blocking PHA stimulated T-cell division.

Various antibodies have been tested with respect to their ability to block PHA stimulated T-cell division, as described previously / Krensky et al., J. Immunol. 131: 611-616 / 1983 //.

Figure 10. Effect of anti-ICAM antibodies on mixed lymphocyte reaction.

Various antibodies have been tested for their ability to block mixed lymphocyte response as described previously. / Krensky et al., J. Ummunol. 131: 611-616 / 1983 //.

Figure 11. Gas chromatography of peptide fragments of 1AM-3.

1CAM-3 was purified and degraded with Lys-C. The peptide fragments were separated by high resolution liquid chromatography.

Figure 12. Branch diagram representation 11.2.

The diagram representing clone 11.2 shows the position of NK-10 and NK-17 peptides, the different DNA sequences obtained, and the transmembrane regions.

Figure 13. Sequential comparison of NK-10 primer sequence with human ICAM-1.

The GAP program is used to identify the sequential homology of the NK-10 primer sequence and human ICAM-1 protein.

Figure 14. Sequential comparison of the RM13 primer sequence with human ICAM-1.

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

Figure 15. Sequential comparison of the RN113 primer sequence with human ICAM-2.

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

Figure 16. Sequential comparison of the T7 primer sequence with human ICAM-1.

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

Figure 17. Sequential comparison of the T7 primer sequence with human ICAM-2.

The GAP program was used to identify the sequence homology of the T7 primer sequence and human 1AM-2.

Figure 18. Partial alignment of ICAM-3.

The DNA sequence of clone 11.2 was determined using standard methods.

A. Sequences obtained from the 5'end of ICAM-3. They are obtained by the effect of clone 11.2 with the T7 primer.

B. Sequences derived from the interior of the 1AM-3 gene. These sequences were obtained by acting on clone 11.2 with the NK-10 probe.

C. Sequences obtained from the 3 ends of 1AM-3. The sequences were obtained by acting on clone 11.2 with the reversible RM13 primer.

Description of preferred embodiments of the invention

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

I. LFA-1.

The leukocyte adhesion molecule LFA-1 mediates a wide range of leukocytes, monocytes, natural killer cells, and granulocyte interactions with other cells in immunity and inflammation / Springer, T.A. et al., Ann.Rev.Immunol. 5: 223-252 / 1987 //.

LFA-1 is the receptor for ICAM-1, 1CAM-2 and the newly identified ICAM-3 (disclosed herein). These surface molecules are constitutively expressed in some tissues and induced in others by inflammation / Marlin, S.D. et al., Cell 51: 813-819 (1987); Dustin, M.L. et al., Immunol.Today 9: 213-215 (1988); U.S. Patent Appl.Ser. No. 07/019 440, 02/26/87 and U.S. Pat. Appl.Ser. No. 07/250446, claimed 28.09.88 р.

LFA functions simultaneously as an antigen-independent and antigen-specific Tcytotoxic, T-helper, naturally-killing, granulocytic and monocytic in its interactions with other cell types / Springer, T.A. et al., Ann.Rev.Immunol. 5: 223-252 (1987); Kishimoto, T.K. et al., Adv.Immunol. (1988, under seal).

P. ICAM-1

ICAM-1 is a single-stranded glycoprotein, varying in mass across cell types of 76-114 kD, and is a member of the Ig superfamily with five C-like regions (Dustin, MLet al., Immunol. Today 9: 213-215 / 1988). ; Staunton, D.E., et al .. Cell 52: 925-933 / 1988 /; Simons, D. et al .. Nature 331: 624-627 (1988). ICAM-1 is highly inductive with cytokines, including IFN-g, TNF, and 1L-1 in a wide variety of cell types (Dustin, M. L. et al., Immunol. Today 9: 213-215 (1988). Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1 dependent adhesion of lymphocytes / Dustin et al.I. Immunol. 137: 245-254 (1986); Dustin et al., J. Cell.Biol. 107: 321-331 / 1988 ?; Dustin et al., J.Exp.Med. 167: 1323-1340 (1988). Adhesion is blocked by re-treating lymphocytes with LFA-1 Mav or re-treating other cells by ICAM-1 MA _in / Dustin, MLet al., J.Exp.Med. 167: 1323-1340 / 1988 //. Identical results with purified ICAM-1 in artificial membranes or on petri dishes indicate that LFA-1 and ICAM-1 are receptors for another (Marlin, SD et al., Cell 51: 813-819 (1987); Makgoba, MWet al., Nature 331: 86-88 / 1988 //. For clarity, they are referred to herein as "receptors" or "ligands". A further description of ICAM-1 is provided in USPat.Appel. No. 07 / 045963,07 / 115 798, 07/155943, 07/189 815 or 07/250 446.

III. 1SAM-2.

Other ligands other than ICAM-1, which are also LFA-1, are accepted. / Rothlei, R. et al., J. Immunol. 137: 1270-1274 (1986); Makgoba, M.W. et al., Eur. J. Immunol. 18: 637-640 (1988), Dustin et al., J. Cell.Biol. 107: 321-331 (1988). The second identified LFA-1 ligand is designated "ICAM-2".

ICAM-2 differs from ICAM-1 in cellular distribution and lack of cytokinin induction. ICAM-2 is a complete membrane protein with two Jg-like regions, while ICAM-1 has 5 Ig-like regions /Staunton, D.E. et al., Cell 52: 925-933 (1988); Simons, D.et al., Nature 331: 624-627 / 1988 //. In short, ICAM-2 is slightly more bound to the two N-terminal regions of ICAM-1 (34% identity) than ICAM-1 or ICAM-2 are to the other members of the Jg-superfamily, exhibiting a subfamily of Jg-like ligands. that bind the same integrin family receptors. A further description of ICAM-2 is given in U.S. Pat. patent description No. 07/454 294.

IV. ICAM-3.

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

The development of MA _in towards ICAM-2 allows the analysis of LFA-1-dependent, ICAM-1-independent phenomena and the assumption that a third ligand for LFA-1 exists. The binding of several cell types, such as epithelial and endothelial cells to purified LFA-1, can be completely blocked by a combination of ICAM-1 and ICAM-2 MA _in which the ICAM-1, ICAM-2-independent adhesion pathway to LFA -1 exists in many lymphoid cell lines, including SKW3 / FIG. 1 /.

To characterize the mode of adhesion, MA _c are cultured to SKW3 and, respectively, with anti-ICAM-1 and anti-ICAM-2 MA _c , screened for their ability to inhibit, binding this cell line to purified LFA-1. One MA _in , CBP-IC3 / 1 was selected, which completely inhibits this new adhesion pathway / FIG. 1 /. SKW3 adhesion to purified LFA-1 is only marginally inhibited by the combination of blocking antiICAM-1 and anti-1CAM-2 MA. When antiV

ICAM-3 MA _in , CB-IC3 / 1 are added alone, adhesion is noticeably inhibited and inhibition is complete when combined with blocking anti-1 CAM-2 MA _in . Thus, adhesion of SKW3 to purified LFA-1 is mediated longer than ICAM-3 and also, in part, by ICAM-2. When adhesion to LFA-1, each of the four cell lines utilized the three ICAM in different degrees, as demonstrated by a variety of samples of _MAb inhibition. The adhesion of the B lymphoblastoid cell line, JY, occurs primarily through the ICAM-1 pathway with little assistance from the ICAM-2 and ICAM-3 pathways. Another In the lymphoblastoid cell line, SLA used concurrently with ICAM-1 and ICAM-3, insofar as adhesion is not inhibited by either ICAM-1 or ICAM-3 MA alone and

In almost completely from MA _in together. The thymic cell line, Jurkat, uses both ICAM-2 and ICAM-3 adhesion pathways with little assistance from 1AM-1. The adhesion of both T-lymphocytes, those at rest and those activated by phytohmagglutin (PHA), was also investigated (FIG. 1B /. Resting lymphocytes have previously been shown to bind tightly to purified LFA-1 (Dustin et al., Nature 341: 619624 (1989), and this binding has been found to be highly ICAM-3 dependent. Upon activation with PHA, ICAM-1 expression is induced and adhesion to LFA-1 occurs mainly through ICAM-1 and partly through ICAM-3. The ICAM-2 component of adhesion in both resting cells and activated T cells is detected, although masked by the presence of ICAM-1 and ICAM-3. There is comparative excess in the use of ICAM for each cell, ICAM monoclonal antibodies are required for at least two ICAMs to achieve significant inhibition. An important exception to this is the adhesion of resting lymphocytes to LFA-1, which are widely mediated. In all cases, the combination of all three anti-1CAM-MA _in eliminates LFA-1 binding to levels comparable to those observed in the presence of anti-LFA-1 MA.

IN

The comparative affinity for the three ICAMs for LFA-1 can be tested by comparing their assistance for LFA-1 binding, as measured above for their cell surface expression as determined by immunofluorescence cytometry / FIG. 2 /. Comparison of the surface expression of ICAM and their contribution to LFA-1 adhesion shows that ICAM-1 has greater affinity for LFA-1 and that ICAM-2 and ICAM-3 have similar but lower affinities. For example, Jurkat cells express ICAM-2 and ICAM-3 at similar levels, and each promotes LFA-1 binding. When ICAM-2 expression is greater than that of ICAM-3, as in IY cells, the ICAM-2 pathway of LFA-1 adhesion dominates that of ICAM-3. In contrast, SLA and SKW3 express three times more ICAM-3 than ICAM-2, and the ICAM-3 adhesion pathway predominates over that of ICAM-2. In resting T lymphocyte cells, ICAM-3 is well expressed and ICAM-1 is lacking, adhesion with LFA-1 is mediated mainly by ICAM-3. When ICAM-1 is well expressed, as is the case for SLA, IY, and PHA-activated T cells, this is the primary / major / pathway of adhesion.

The distribution pattern of ICAM-3 differs from that of ICAM-1 and ICAM-2. Unlike ICAM-1 and ICAM-2, ICAM-3 is not expressed in resting or stimulated endothelial cells (data not shown). This is inconsistent with the finding that LFA-1-dependent cell binding to resting or activated endothelial cells is an ICAM-1 and ICAM-2-dependent phenomenon. ICAM-3 is restricted to the hematopoietic lineage and is highly expressed in lymphoid and monocytic cell lines, with few exceptions. In all cases, however, the expression of ICAM-3 in the cell lines is coordinated by the LFA-1-dependent, ICAM-1, ICAM-2-independent adhesion mode. Cell lines (HUVEC, Raji) binding LFA-1 solely through ICAM-1 and ICAM-2 do not show ICAM-3 expression, whereas cell lines (JY, U937, Sup T) that show weak binding through these three adhesion pathways have a corresponding low ICAM-3 surface expression (data not shown).

1C A.M-3 are significantly different from ICAM1 and ICAM-2 in their leukocyte expression / FIG. 2B /. ICAM-3 is expressed at high levels in resting cell lymphocytes, monocytes, and neutrophils, while ICAM-1 and ICAM-2 are slightly less or absent. In comparison, ICAM-1 and ICAM-2 are less expressed in monocytes, and only ICAM-2 is present in quiescent cells. Neither ICAM-1 nor ICAM-2 is expressed in neutrophils. Upon activation of lymphocytes with PHA, ICAM-3 expression was increased 2–3-fold, whereas ICAM-1 expression was strongly induced / Dustin et al., J. Immunol. 137: 245254/1956 // FIG. 2B /.

Immunoprecipitates of ICAM-3 from different '-Ί labeled cell .linii revealed a sharp band of 124 000M _d in re people healing conditions with only slightly increased mobility under nonreducing conditions / Fig. FOR /. Treatment with N-glycanase reduced the ICAM-3 band to M _g 87000, indicating that ICAM-3 was similar to ICAM-1 and ICAM-2. is a highly glycosylated protein / FIG. SV /. The biochemical characteristics, mode of expression and functional properties of ICAM3 distinguish them from previously described adhesion molecules, including the human homingreceptor LAM-1 / Tedder et al., J. Immunol. 1.144: 532540 (1990), the inducible endothelial adhesion molecule VACAM-1 (Rice et al., J.Exp.Med. 171: 1369-1374 (1990); Carlos et al., Blood and Press / 1990 /; Osborn et al., Cell 59: 1203-1211 / 1989 // and the VLA family of matrix receptors / Hemler, ME, Ann, Rev. Immunol.8: 365-400 (1990). with no MA _in similar cellular distribution / Cilks, W, R. et al., Leukocyte Typing Data Base IV, Oxford University Press, Oxford, England, / 1990 //.

The existence of three LFA-1 ligands implies specialization of various aspects of LFA-1-dependent interactions. 1CAM-1 is mainly expressed in the endothelium and in many epithelial cell types and is highly induced in inflammation and immunity, where it regulates cellular localization and facilitates specific antigen recognition. / Wawryk et al., Immunol.Rev. 108: 135161 (1989). Insofar as ICAM-2 is a predominant LFA receptor, this mode of adhesion may have significant consequences for the normal recycling of LFA-1-bearing lymphocytes through tissue endothelium (Hamman et al., J. Immunol 140: 639-699 (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.Pth. 19: 753-759 (1988). At present, the functions of ICAM-3 on neutrophils remain unclear, since the homotypic aggregation of neutrophils is primarily Mas-1 dependent, due to the presence of LFA-1 / Anderson et al., J. Immunol. 137: 15-27 (1986); Patarrovo et al., Scand. J.Immunol. 22: 619-631 / 1985 //. The finding that adhesion of resting T lymphocytes to LFA-1 is primarily accomplished by ICAM-3, in combination with the fact that ICAM-3 is better expressed than other LFA-1 ligands on monocytes and located in resting lymphocytes play an important role in the initiation of immune responses. In fact, T-cell adhesion with antigen-delivering cells requires LFA-1: ICAM interactions / Dransfield et al., Immunol.Rev. 114: 29-44 / 1990 /: Makgoba et al., Immunol.Today 10: 417-422 (1988), and as such, ICAM-3 may play a role, especially in resting T lymphocytes, where neither ICAM-1 nor ICAM-2 are well expressed. Indeed, such a role has been suggested for LFA-1 ligands other than ICAM-1 as well as allogeneic and autologous mixed lymphocyte responses / Bagnasco et al., Cell Immunol. 128: 326-369 (1990). Something more. ICAM-3 may be suggested to be important in the antigen-specific interactions between T- and B-lymphocytes, where one of these cells has not yet been activated. ICAM-3 may also be important in lysis of important T-cell targets, which is dependent on LFA-1 but not on ICAM-1 / Makgoba et al., Eur.J.Immunol. 18: 637-640 (1880).

The existence of multiple ICAMs has important applications for clinical treatment of MA _in relation to ICAMs or ICAM analogues. ICAM-1 MA _c is fixed in vivo in prolonged kidney / Cosimi et al., J. Immunol. 144: 4604-4612 (1990) and cardiac / Flavin et al., Transpl. Proc. 23: 533534/1991 / Allografts. 1CAM-3 MA _b inhibits distinguished and overlapping immune responses in vivo insofar as it inhibits LFA-1-dependent interactions of distinguished cell type subsystems.

V. cDNA Cloning of ICAM-3

Any of the known techniques may be used to clone the ICAM3 gene. One such method involves analyzing a library of shuttle vectors of cDNA inserts (derived from ICAM-3 expression cells) for the presence of an insert containing the ICAM-3 gene. Such analysis can be performed by transfection of cells with a ventricle and then an ICAM-3 expression assay.

ICAM-3 cDNA is preferably identified using a modified method of Arruffo and Seed / Seed, B. et al., Proc.Natl. Acad.Sci. USA 84: 3365-3369 (1987) to identify ligands from adhesion molecules. In this method, a cDNA library is made from cells that express ICAM-3 (such as SLA, Jurkat, or SKW3 lymphoblastoid cell lines). Preferably, the cDNA library is prepared from T cells. This library is used for transfection of cells that do not normally express ICAM-3 (such as Cos or HeLa cells). The transfected cells were introduced into petri dishes that were pre-coated with LFA-1 or anti-ICAM-3 antibodies. Transfected cells containing ICAM-3 coding sequences, and which express these ligands on their cell surface, will adhere to LFA-1 or anti-1CAM-3 antibodies from the surface of the petri dishes. The non-adherent / non-adhering / cells are washed, and those adherent are removed and cultured. The recombinant ICAM-3 expression sequences in these cells are then removed and matched.

If LFA-1 is used to cover the petri dish, anti-ICAM-1 and anti-ICAM-2 specific antibodies are added to prevent adherence of ICAM-1 or ICAM-2 expressing cells. The adherence of ICAM-1 *, or ICAM-2 + transfectants to LFA-1 with the plates can be inhibited thereby to antibodies such as RR1 / 1, an anti-ICAM1-1 MAb _and CBR-1C _2/2, anti- 1SAM-2

MA _c . The binding of ICAM-3 transfected cells to LFA-1 coated plates was inhibited by EDTA and anti-LFA-1 MA _bs but was not inhibited by anti-ICAM-1 or anti-1CAM-2 MA _m . Therefore, ICAM-1 or ICAM-2 expression cells are unable to adhere to the petri dishes and are therefore able to be flushed with all other nonadherent cells. This is enriched in cells expressing ICAM-3.

Other alternative methods for producing sequences of the gene encoding ICAM-3 are well known to those skilled in the art and can be readily adapted to produce the desired gene.

One such method of producing a gene sequence that encodes ICAM-3 is one that uses an oligonucleotide probe to screen a cDNA or genomic DNA library. In this method, the ICAM-3 protein is purified using known immuno-purification techniques, and the final amino acid sequences are determined using one of the methods known in the art. Conversely, ICAM-3 enzymatically purified (either with trypsin or Lys-C) peptides were purified, and the amino acid sequence of one of the internal fragments was determined.

Once a partial amino acid sequence has been determined, an oligonucleotide probe is created based on the colon preferences shown by the body, a degenerate probe is created based on all possible codon combinations or codon preference combinations homologous to known ICAM-1 or ICAM-1 or ICAM sequences, and codon degeneration is used to construct probes. This probe is then used to screen both the genomic and cDNA libraries for sequences that hybridize to the probe.

Methods such as or analogous to those described above are capable of substantially cloning human aldehyde dehydrogenase genes / Hsu, L.C. et al., Proc. Natl. Acad.Sci.USA 82: 3771-3775 / 1985 //, fibronectin / Suzuki, S.et al., Eur. Mol.Biol. OrganJ. 4: 2519-2524 (1985), human estrogen receptor gene / Walter P.et al., Proc. Natl. Acad. Sci.USA 82: 7889-7893 (1985), Tissue Human Plasminogen Activator (Pennica D. et al., Nature 301: 214-221 / 1983).

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

The cloned ICAM-3 gene obtained by any of the methods described above can be operatively attached to an expression vector and introduced into a bacterial or eukaryotic cell to produce the ICAM-3 protein. Techniques for such manipulations have been disclosed by Maniatis, T., et al., Supra, and are well known in the art.

In an alternative method using PCR for cloning into the ICAM-3 gene, the assumption is made that ICAM-3 is homologous to ICAM1 and / or ICAM-2. Sequence-based degenerate oligonucleotide primers are used. stored between ICAM-1 and ICAM2 amplified by polymerase chain reaction cDNA or mRNA from cells known as expressing ICAM-3 protein, for example SKW3 or tonsil / deFougelle et al., J. Exp.Med., 175.185-190 / 1992 /. The clones were matched and those different from ICAM-1 and ICAM-2 were used to produce full-length cDNA clones.

The authenticity of clones obtained by any of the methods can be confirmed by expression of full-length clones, for example, in COS cells, and tested for reactivity with 1AM-3 mAv.

VI. Factors of the present invention: ICAM-3 and its functional derivatives, agonists and antagonists

The present invention is directed to ICAM-3, its " functional derivatives ", its agonists " and " antagonists ".

A. Functional Derivatives of ICAM-3 The "functional derivative" of ICAM-3 is one that has a biological activity (both functional and structural) that is substantially analogous to the biological activity of ICAM-3. The term "functional derivative" includes "fragments", "variants" and "chemical derivatives" of the parent 1AM-2 molecule.

The ICAM-3 "fragment" is used to refer to any molecule subsystem. ICAM-3 fragments that have ICAM-3 activity and are soluble / i.e. not membrane-bound / are particularly preferred. The soluble fragment is preferably obtained by separating the regions associated with membrane bridges from the parent molecule, or by replacing the hydrophilic amino acid residues with hydrophobic residues. The identification of such residues is known in the art. Fragments containing any number from the entire Jg-like region, such as region 1, (E1), E1 and E2, E1-3, E1-4 and E1-5, are preferred.

An "variant" of ICAM-3 means that it refers to a molecule essentially of similar structure and function, with both the entire molecule and its fragment.

One molecule is said to be "substantially analogous" to another molecule if the two have substantially similar structures or if they achieve similar biological activity. Thus, if two molecules attain similar biological activity, they are considered variants as used in the present text, even if one of them has a structure that is not found in the other molecule, or if the sequence of amino acid residues is not identical.

As used herein, one molecule is said to be a "chemical derivative" of another molecule when it contains additional amounts that are not normally part of a naturally occurring molecule. Such amounts can improve molecular solubility, adsorption, biological half-life, etc. The amount can also counteract the toxicity of the molecule or eliminate or attenuate some of the unwanted side effects. Amounts capable of mediating such effects are described in Remington Pharmaceutical Science (1980).

"Toxin derivatives" are a special class of "chemical derivatives". "Toxin derivatives" are molecules (such as ICAM-3 or anti-ICAM-3 antibody) that are covalently attached to any amount of toxin. Procedures for coupling such amounts to a molecule are well known in the art.

The binding of such a molecule to a cell brings the amount of toxin in close proximity to the cell and therefore leads to cell division. However, any suitable toxin amount may be used, however, preferably such as castor, cholera, diphtheria toxin, radioisotope toxin or membrane-forming toxins.

Functional derivatives of ICAM-3 having up to about 100 residues can be readily prepared by in vitro synthesis. Optionally, such fragments can be modified by methods known in the art in reaction of a target amino acid residue from a purified or crude protein with an organic derivative capable of reacting with a selected side chain or with terminal residues. The resulting covalent derivatives can be used to identify residues important for biological activity.

Many different methods can be used to produce and isolate fragments of ICAM-3. The preparation of derivatives with bifunctional factors can be used to cross-link 1AM-3 to a water-insoluble matrix. In contrast, reactive, water-insoluble matrices, such as cyanogen-bromo-activated carbohydrates and reactive substrates, described in US Nos. 3969287, 3691016, 4195128, 4247642, 4229537 and 4330440 are used to immobilize the protein.

Functional derivatives of ICAM-3 having altered amino acid sequences can also be prepared by mutation of DNA encoding ICAM-3. Such functional derivatives include, for example, deletion of either insertion or substitution into residues within the amino acid sequences of ICAM-3. Any combination may be used to produce the final structure having the desired activity. It is imperative that the mutations that will be carried out in the DNA encoding the functional derivatives do not place the sequences outside the reading frame and preferably do not create complementary regions that produce a secondary mRNA structure (see EP No. 75 444).

At the genetic level, functional derivatives can be generated by site-directed mutagenesis for DNA encoding ICAM-3, and then DNA is obtained encoding functional derivatives and subsequently expressing DNA in recombinant cell culture.

While the insertion site of the amino acid sequence variant is predetermined, the mutation per se does not need to be predetermined. For example, to optimize a site mutation, random mutagenesis, such as a linker, can be performed at a target codon or target region to generate a large number of derivatives that can then be expressed and screened for optimal combination with the desired activity. Such techniques that allow mutations at predetermined sites of DNA are well known, for example, site-directed mutagenesis.

The preparation of functional derivatives of ICAM-3, as described above, was achieved by site-directed mutagenesis of DNA encoding ICAM-3 or an earlier derived derivative of ICAM-3. The technique of such site-specific mutagenesis is well known in the art, as can be seen in the publications of Maniatis, T. et al., In: Molecular Cloning, a Lab Manual, Coldspring Harbor, NY / 1982 /. Site-directed mutagenesis allows the production of ICAM-3 functional derivatives by using specific oligonucleotide sequences that encode a DNA sequence with the desired mutation.

Some may generate amino acid deletions, insertions or substitutions through site-directed mutagenesis. Amino acid deletions generally range from about 1 to 30 residues, preferably 10 to 1 residues, and are usually adjacent. The most preferred deletions are those that generate soluble form of ICAM-3. Soluble forms of ICAM-3 are generated mainly by deletion of both ICAM-3 membrane bridges and hydrophobic residues within ICAM-3.

Amino acid insertions include insertions of single or multiple amino acid residues within ICAM-3 coding sequences such as terminal fusions with substantially unlimited length polypeptides. Intra-sequential insertions / i.e.

insertions within the complete ICAM-3 molecular sequence / may mainly border from about 1 to 10 residues, preferably from 1 to 5. An example of a terminal insertion involves a fusion of a signal sequence, whether heterologous or homologous to the host cell, with the N-terminus of the molecule to facilitate the secretion of the derivatives by the recombinant host.

The third group of functional derivatives are those in which at one or more amino acids in the ICAM-3 molecule the residues are removed and various other residues are inserted in their place. Such substitutions are preferably made in accordance with the following table when it is desirable to modify the characteristic of the ICAM-3 molecule.

Table 1

Original acid residue Sample substitutions Ala gly: Ser Arg lys Asn gln: his Asp glu Cys sir Gin asn Glu asp Gly ala: pro His asn: gln lie leu: Val Leu ile: Val Lys arg: gln: glu Met leu: tyr: ile Phe meth: leu: tyr Sir thr Thr sir Trp tyr Tyr trp: phe Vai ile: leu

Substantial changes in functional or immunological identity were made by selecting substitutions that were less conservative than those shown in Table 1, i. E. picking up residues that are more significant in their storage effects:

a) the structure of the polypeptide skeleton in the substitution region, for example as a broad strip or a helicoidal structure;

B) the charge or hydrophobicity of the molecule at the target site, or c / the bulk of the side chain.

The substitutions that are generally less conservative are those in which:

a) glycine and / or proline are substituted by another amino acid or are deleted or inserted;

B / the hydrophilic residue is substituted with hydrophobic;

c / cysteine residue is replaced by any other residue;

d / the electropositive side chain residue is replaced by an electronegative charge residue and the bulky side chain residue is replaced by one that does not have such a side chain.

More preferred are substitutions that affect the solubility of ICAM-3. They are generated by substituting hydrophilic with hydrophobic amino acids.

Mutations designed to enhance the affinity of ICAM-3 can be driven by introducing the amino acid residues present at homologous positions in ICAM-1 or ICAM-2. Thus, such mutant ICAM-3 molecules can be created in which there is no N-linked CHO at homologous positions in ICAM-1 or ICAM-2.

It is difficult to predict what effect a substitution, deletion or insertion would have on the biological activity of ICAM-3. But one skilled in the art would be able to evaluate the effect through routine research. For example, a derivative is usually obtained by site-directed mutagenesis of DNA encoding a native ICAM-3 molecule. The derivative is then expressed in a recombinant host, and can be purified from cell culture, for example by immunoaffinity chromatography.

The activity of the cell lysate or purified derivative is then screened in suitable screening assays for the desired characteristics. For example, the change in the immunological nature of functional derivatives, such as affinity for an antibody, is measured by con17 cient type of immunoassays. Changes in immunomodulatory activity are measured by appropriate methods. Modifications of such protein properties as redox or thermostability, biological half-life, hydrophobicity, susceptibility to protein degradation, or tendency to aggregate with carriers or in multimers are tested by well-known methods in the art.

B. ICAM-3 agonists and antagonists

An ICAM-3 agonist is a compound that enhances or enhances the ability of ICAM-3 to perform any of its biological functions. An example of an agonist is one factor that enhances the ability of ICAM-3 to bind to cellular receptors.

An ICAM-3 antagonist is a compound that attenuates or impedes the ability of ICAM-3 to perform any of its biological functions. Examples of such antagonists include ICAM-1 and ICAM-2, functional derivatives of ICAM-1 and ICAM-2, anti-ICAM-3 antibody, anti-LFA-1 antibody, and the like.

Cell aggregation assays described in US 07/045963 and 07/115943, 07/189815 or 07/250446 show the possibility of measuring LFA-1 dependent aggregation and thus can be used to identify the factors that influence the prolongation of ICAM-3 / LFA-1 aggregation. Thus, such studies can be used to identify agonists and antagonists of ICAM-3. Antagonists may act by impairing the ability of LFA-1 or ICAM-3 to mediate aggregation. In addition, non-immunoglobulin / i.e. chemical / agents can be tested using the above test methods to determine whether they are agonists or antagonists of 1AM-3 / LFA-mediated aggregation. Such aggregation assays are typically represented by PMA-stimulated peripheral T cells. Thus, binding of peripheral blood cells to LFA-1 is used to investigate ICAM-3 mediated aggregation.

C. Anti-1AM-3 antibody

The preferred immunoglobulin antagonist of the present invention is an antibody against ICAM-3, for example CBR-JC3 / 1 described herein. Other suitable antibodies may be prepared by any of the known methods.

The hybridoma cells described and prepared as described above can be screened by various methods to identify a hybridoma cell secreting an antibody capable of binding ICAM-3. In the preferred screening method, such molecules are identified by their ability to inhibit the aggregation of ICAM-3 expressing, ICAM-1 and ICAM-2 non-expressing cells. Antibodies that are capable of inhibiting such aggregation are further tested to determine whether they inhibit aggregation by joining ICAM-3 or by attaching to any member of the CD-18 family of molecules. Any agent capable of distinguishing ICAM-3 from the CD-18 family of molecules may be used in this study. For example, the antibody bound by the antibody can be analyzed both by immunoprecipitation and by polyacrylamide gel electrophoresis. It is possible to distinguish between those antibodies that bind to members of the CD-18 family and those that bind ICAM-3 by screening for the antibody's ability to bind to cells expressing LFA-1 but not ICAM -With / but not Vice Versa /. The ability of an antibody to bind to a cell expressing LFA-1 but not ICAM-3 can be established by agents widely used by one of skill in the art. Such agents are: immunoassays (in particular those using immunofluorescence), cellular glutination, antibody precipitation, and the like.

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

In addition to the ICAM-3 agonists and antagonists described above and in accordance with the present invention, for the treatment of inflammation, HIV infections, asthma and the like include anti-idiotypic antibodies against anti-ICAM-3 antibodies and receptor molecules or fragments thereof, which are capable of binding to ICAM-3.

The anti-idiotypic antibodies of the invention are able to bind competitively with / or with the exception of / ICAM-3. Such bodies can be obtained, for example, by culturing an antibody against an anti-ICAM-3 antibody and then screening it for its ability to bind to one of ICAM-3's natural binding ligands.

To the extent that the molecules of the CD family are able to bind to ICAM-3, the introduction of such molecules / eg as heterodimers having both a and β subunits, or as molecules composed only of α or β subunits, or as molecules , which have fragments of either or both subunits, will compete with the cells to bind to ICAM-3 on their surface.

The anti-aggregation antibodies of the present invention can be identified and titrated in many different ways. For example, the ability of differentiated binding antibodies to cells that express 1CAM-3 (such as T cells) and their inability to bind to cells that cannot express 1CAM-3 can be measured. Suitable cell aggregation assays are described in US Nos. 07/045963, 07/115798, 07/115943, 07/189815 or 07/250446. Conversely, the capacity of antibodies to bind to ICAM-3 or to a peptide fragment of ICAM-3 can be measured. As can be established by one of ordinary skill in the art, the methods described above can be modified or presented in different sequences to provide a variety of screening techniques, each capable of identifying and distinguishing individual antibodies capable of binding ICAM- 3 against members of the molecule family designated CD-18.

D. Preparation of Agents of the Present Invention

The factors according to the present invention can be obtained by: natural processes / for example, by the participation of animals, plants, fungi, bacteria, etc. in the production of a non-immunoglobulin antagonist of ICAM-3, or by the production by animals of polyclonal antibodies capable of binding to ICAM-3 /; by synthetic methods (for example, by synthesizing ICAM-3, functional derivatives of ICAM-3, or ICAM-3 protein antagonists (both immunoglobulin and non-immunoglobulin); by means of a hybridoma technique (eg, for the production of monoclonal antibodies capable of binding to ICAM-3) or by recombinant technology, such as for the preparation of the agents of the present invention in various hosts (such as yeast, bacteria, fungi, cultured mammalian cells and etc. / using recombinant plasmids or viral vectors /. The choice of method used will depend on factors such as convenience, desired yield, etc. However, it is not necessary to use only one of the described methods, processes and technologies for the preparation of a special anti-inflammatory agent, they can be combined to produce a specific agent.

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

The agents of the present invention can be used to mitigate various biological activities mediated by ICAM-3.

A. Suppression of inflammation

An aspect of the present invention stems from the ability of ICAM-3 and its functional derivatives to interact with the receptors of the CD-18 molecular family of glycoproteins, in particular LFA-1. Through the ability of ICAM-3 to interact with members of the CD-18 family, it can be used to suppress / ie. to prevent or weaken / inflammatory processes.

The term "inflammation" as used herein means that it includes the reactions of both the specific and the non-specific defense system.

A specific defense system refers to that component of the immune system that responds to the presence of specific antigens. Inflammation is considered to be the result of a response to a specific defense system if the inflammation is caused by, mediated by, or associated with the specific defense system. Examples of inflammation resulting from a response to a specific defense system include the response of antigens such as rubella virus, autoimmune diseases, and the delayed type of cell-mediated hypersensitive response as seen, for example, in individuals who respond positively to the

Mappaich. Chronic inflammatory diseases and the rejection of transplanted tissues and organs, such as the kidneys and bone marrow, are other examples of inflammatory responses to a specific defense system.

The response of the nonspecific defense system is mediated by leukocytes that are incapable of immunological memory. Such cells include granulocytes and macrophages. Inflammation is considered to be the result of a response to a non-specific defense system if it is caused by, mediated by, or associated with a response of a non-specific defense system. Examples of inflammation that lead, albeit in part, to a response of a non-specific defense system include inflammation associated with conditions such as Age Respiratory Imbalance Syndrome (ARDS) or multiple organ damage, secondary to septicemia, trauma, or hemorrhage; reperfusion damage to the myocardial or other tissues; acute glomeronephrosis; reactive arthritis; dermatoses with acutely inflammatory components; acute purulent meningitis or other inflammatory disorders of the central nervous system such as stroke, thermal injury, hemodialysis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis; associated granulocyte transfusion syndrome and cytokinininduced toxicity.

As discussed, the binding of ICAM-3 molecules to members of the CD-18 molecular family is central to cell adhesion. Through the adhesion process, lymphocytes are capable of continuously monitoring and, more precisely, controlling an animal for the presence of foreign antigens. Although these processes are normally desired, they are also the cause of organ transplant rejection (such as kidney, bone marrow) and tissue transplants, as well as many autoimmune diseases. Thus, any agent that is able to delay or inhibit cell adhesion would be highly desirable for recipients of solid organs (and in particular renal transplants), soft organs (especially bone marrow), tissue transplants, or patients with autoimmune diseases.

Monoclonal antibodies against members of the CD-18 family inhibit many adhesion-dependent leukocyte functions, including binding to the endothelium / Haskard, D. et al., J. Immunol. 137: 2901-2906 / 1986 //, homotypic adhesion / Rothlien, R. et al., J.Exp.Med. 163: 1132-1149 / 1986 //, antigens mitogen-induced lymphocyte proliferation / Davignon.D. et al., Proc.Natl.Acad.Sci., USA 78: 4535-4539 / 1981 //, antibody formation / Ficher, A. et al., J. Immunol. 136: 3198-3203 / 1986 // and the effector functions of all lymphocytes as lytic activity of cytotoxic T cells /Krensky, A.M. et al., J. Immunol. 132: 2180-2182 / 1984 //, macrophages / Strassman, G. et al., J. Immunol. 136: 4328-4333 / 1986 //, and all cells involved in antibody-dependent cytotoxicity reactions / Kohl, S., et al., J. Immunol. 133: 2972-2972 (1984). In all of the above functions, the antibodies inhibit the ability of leukocytes to adhere to suitable cellular substrates, which in turn inhibit the biological functions associated with binding. Such functions, as continued to include ICAM-3 / LFA-1 interactions, can be suppressed with antiICAM-3 antibodies.

Thus, monoclonal antibodies capable of binding to ICAM-3 can be used as an anti-inflammatory agent in mammals. Remarkably, these agents differ from the anti-inflammatory agents in that they are capable of selective inhibitory adhesion and show no other side effects, such as the nephrotoxicity found with conventional agents.

To the extent that ICAM-3, in particular in soluble form, is capable of acting in the same way as an antibody against members of the CD-18 family, it can be used to suppress inflammation. Moreover, functional derivatives and antagonists of ICAM-3 can also be used to suppress inflammation.

1. Suppressors of delayed hypersensitivity reactions

ICAM-3 partially mediates the adhesion events required to build inflammatory responses, such as that of delayed hypersensitivity. Thus, antibodies (especially monoclonal antibodies) that are capable of binding ICAM-3 have therapeutic potential in delaying or eliminating such reactions.

Conversely, insofar as ICAM-3 is an antagonist of the ICAM-1 (LFA-1) interaction, ICAM-3 / in particular in soluble form: or its functional derivatives can be used to suppress delayed-type hypersensitivity reactions.

These potential therapeutic options can be used in two ways. First, a composition comprising an anti-ICAM-3 monoclonal antibody, or soluble derivatives of ICAM-3, may be administered to patients suffering from a delayed-type hypersensitivity reaction. For example, such compositions may be administered to humans who have been in contact with antigens of certain types of ivy and oak. In other cases, a monoclonal antibody capable of binding to ICAM-3 is administered to patients who are in contact with antigens to prevent the last inflammatory response. Thus, the additional administration of an antigen with an anti-ICAM-3 antibody may act to temporarily tolerate the individual to the subsequent presence of the given antigen.

2. Treatment of chronic inflammatory disease.

To the extent that LAD patients who do not have LFA-1 do not construct an inflammatory response, it is thought that antagonism of LFA-1 ligands will also inhibit the inflammatory response. The ability of antibodies against ICAM-3 to inhibit inflammation underlies their use as therapeutic agents against inflammatory and autoimmune diseases such as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, multiple sclerosis, rheumatoid arthritis, etc. Such antibodies can also be used to treat psoriasis.

In general, the anti-ICAM-3 antibodies of the present invention may be administered alone or in combination with anti-ICAM-1 and / or anti-ICAM-2 antibodies in the treatment of those diseases which have recently been treated with steroid therapy.

According to the present invention, such inflammatory or immune rejection responses can be suppressed (i.e., suppressed) - i.e. both prevented and delayed by providing the patient in need of such treatment with the necessary amount of anti-inflammatory agent sufficient to suppress inflammation. Suitable anti-inflammatory agents are: an antibody capable of binding to ICAM-3, a fragment of that antibody capable of binding to ICAM-3; substantially pure ICAM-3; functional derivatives of ICAM-3; non-immunoglobulin antagonist of ICAM-3 other than LFA-1. Particularly preferred are anti-inflammatory agents composed of soluble functional derivatives of ICAM-3. Such anti-inflammatory treatment may also include the additional administration of an agent selected from the group consisting of: an antibody capable of binding to LFA-1; non-immunoglobulin antagonist of LFA-1; substantially pure ICAM-1 and / or ICAM-2 or derivatives thereof, or anti-ICAM-1 and / or anti-ICAM-2, or derivatives or fragments thereof.

The invention includes the described methods for suppressing the inflammatory response by the specific defense system, which provides an additional immunosuppressive agent for the subject of treatment. Such an agent is preferably provided at doses lower / ie. suboptimal doses / of those normally required. Suboptimal dosage administration is possible due to the synergistic effect of the agents of the present invention. Examples of suitable immunosuppressive agents are (but are not limited to) dexamethasone, azathioprine, ICAM-1, ICAM-2, or cyclosporin A,

3. Treatment of nonspecific inflammation Many inflammatory reactions are due to reactions of the nonspecific defense system and are determined by leukocytes incapable of immunological memory. Such cellulites are granulocytes and macrophages. As used herein, inflammation is considered to be the result of a response to a non-specific defense system if the inflammation is caused by, mediated by, or associated with a reaction of the non-specific defense system. Examples of inflammation due, at least in part, to a response to a nonspecific defense system are those associated with adult respiratory distress syndrome or secondary organ disorder syndromes resulting from septicemia, acute glomerulonephritis, reactive arthritis, dermatitis acute inflammatory components, acute purulent meningitis or other disorders of the central nervous system, such as stroke, thermal damage, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, a syndrome associated granulocyte transfusion and cytokinininduced toxicity.

The anti-inflammatory agents of the present invention are compounds capable of specifically resisting the interaction of the CD-18 complex on granulocytes with endothelial cells. Such antagonists include: ICAM-3, functional derivatives of ICAM-3, an immunoglobulin antagonist of ICAM-3 other than ICAM-1, ICAM-2 or members of the CD-18 family.

B. Organ and tissue rejection suppressors

To the extent that 1CAM-3, in particular in soluble form, is capable of acting in the same way as an antibody to the members of the CD-18 molecular family, it can be used to suppress the rejection of organs or tissues caused by either be of cell adhesion-dependent function. Moreover, an anti-ICAM-3 antibody, its functional derivatives and ICAM-3 antagonists can also be used to suppress this reaction.

ICAM-3 and anti-ICAM-3 antibodies can be used to prevent organ rejection in transplantation of solid organs such as kidney, soft organs such as bone marrow, or in modified autoimmune responses, for example in mammals. Importantly, the use of a monoclonal antibody capable of recognizing ICAM-3 may allow organ transplantation to occur even between individuals with HLA mismatches.

C. Addition to the antigenic material introduced for therapeutic or diagnostic purposes

Immune responses against therapeutic or diagnostic agents such as bovine insulin, interferon, tissue plasminogen activator, or murine monoclonal antibody significantly impair the therapeutic or diagnostic value of these agents and in some cases may cause serum diseases. Such a situation can be overcome by the use of the antibodies of the present invention. The variant according to the invention provides for anti-ICAM-3 antibodies to be administered in combination with a diagnostic or therapeutic agent. Adding the antibody prevents the recipient from recognizing the appropriate agent and therefore prevents the immune response from being initiated against it. The lack of such an immune response results in the patient's ability to receive additional doses from the therapeutic or diagnostic agent.

ICAM-3, in particular in soluble form, or its functional derivatives may be used alone or in combination with ICAM-1 or ICAM-2, or with antibodies capable of binding to ICAM-1 in the treatment of the disease. Thus, in soluble form, such molecules can be used to inhibit organ or tissue transplantation. ICAM-3 or its functional derivatives can be used in the same way as anti-ICAM-3 antibodies to reduce the immunogenicity of therapeutic or diagnostic agents.

D. Suppressors of tumor metastases

The agents of the present invention can also be used to inhibit hematopoietic tumor cell metastases, which requires the migration of functional members of the CD-18 family. In accordance with the present embodiment, a patient in need of such treatment is provided with an appropriate amount of agent / antibody capable of binding to ICAM-3; a toxin derivative of this antibody; an antibody fragment, the fragment being capable of binding to ICAM-3; a toxin derivative of this fragment; substantially pure 1AM-3; functional derivative of ICAM-3; or a non-immunoglobulin ICAM-3 antagonist other than ICAM-1 or ICAM-2 / sufficient to suppress metastasis.

Further in the embodiment of the present invention there is provided a method of inhibiting the growth of ICAM-3 expressing tumor cells. In particular, this method involves supplying the patient in need with an amount of agent sufficient to suppress the corresponding growth. Suitable agents are: an antibody capable of binding to ICAM-3, a toxin derivative of the antibody; an antibody fragment, such fragment being capable of binding to ICAM-3; a toxin derivative of this fragment, a toxin derivative that is a member of the CD-18 molecular family, or a toxin derivative of a functional derivative of a member of the CD-18 molecular family.

Further, a method of preventing the growth of LFA-1-expressing tumor cells is provided in the embodiment of the invention. In particular, this method involves providing the patient in need of such treatment with an amount of toxin sufficient to suppress said growth. Suitable toxins are toxin derivatives of ICAM-3 or toxin functional derivatives of ICAM-3.

F. Suppression of HIV infection and prevention of the spread of HIV-infected cells.

The present invention further provides a method of suppressing HIV infection. The specificity of this method is that it involves administering to an HIV-infected individual an effective amount of an HIV-suppressing agent. Although the invention relates in part to a method of suppressing HIV-1 infection, it should be understood that this method is applicable to any variant of HIV (such as HIV-2) that can infect cells in a way that can be suppressed by the factors of the present invention. Such variants are the equivalents of HIV-1 for the purposes of the invention.

One aspect of the present invention stems from the recognition that the expression of LFA-1 and, in some cases, HIV-stimulated LFA-1 binding ligands, promotes cell-cell responses to adherence, which may increase the contact time of infected with uninfected cells, facilitating the transfer of virus from infected to uninfected cell. Thus, agents of the present invention that are able to modulate LFA-1 / ligand interactions are able to suppress HIV infection and, in particular, HIV-1 infection. A means by which molecules that inhibit the interaction between LFA-1 and ligands can suppress HIV infection is impairment of the ability of LFA-1 ligands expressed by HIV-infected cells to bind to the T11 / CD18 receptors of healthy T cells. . Conversely, molecules that inhibit LFA-1 / ligand interactions may impair the ability of the CD11 / CD18 receptors expressed by HIV-infected cells to bind to LFA-1 in healthy T cells. In order to impair the ability of a cell to bind to the CD11 / CD18 receptor or to the LFA-1 binding ligand, it may be possible to use ICAM-3, a fragment of ICAM-3, a functional derivative of ICAM-3 or anti-1CAM-3 antibodies.

The factors of the invention are designed to be provided to recipients in an appropriate amount sufficient to suppress HIV infection. An amount is considered sufficient to “suppress” HIV infection if the dose is sufficient to delay or prevent such HIV infection. These factors are available to patients who are at risk of contracting or infected with the HIV virus.

The factors of the present invention can serve both therapeutic and prophylactic needs. When for prophylactic purposes, the agent is provided in advance for any symptom of a viral infection (for example, before, at, or shortly after the time of such infection), but before any symptoms of such infection. Prophylactic administration of the agent serves to prevent or delay any subsequent HIV infection. When used therapeutically, the agent is provided at or shortly after the detection of virus-infected cells. The therapeutic administration of the agent serves to delay any additional HIV infection.

The agents of the present invention can thus be provided as before the onset of a viral infection (to suppress the intended HIV infection) or after the actual detection of such virus-infected cells (to suppress further infection).

Furthermore, the invention provides improved AIDS therapy and improved agents for suppressing HIV infection, in particular HIV-1 infection, which involves the co-administration of:

(I / ICAM-3, or soluble ICAM-3 derivatives, CD11 / as CD11a, CD11c, or CD11c), soluble CD11 derivatives, CD18, soluble CD18 derivatives, or CD11 / CD18 heterodimers, or soluble derivative of CD11 / CD18 heterodimer and / or (II) an antibody capable of binding to ICAM-3 with (III) a cell or a partially associated CD4 or soluble CD4 derivative and / or (IV) a molecule (preferably an antibody or antibody fragment) capable of connect to CD4.

The invention further provides a method of inhibiting the migration of HIV-infected cells. Specifically, the method involves administering an effective amount of an anti-migration agent to an HIV-infected individual.

The antimigration agents of the present invention include ICAM-3, a fragment of ICAM-3, functional derivatives of ICAM-3 or anti-ICAM-3 antibodies, which are able to impair the ability of HIV-infected T cells to bind to LFA- 1-ligand. Antibodies that bind to ICAM-3 will inhibit migration by impairing the ability of ICAM-3 expressed by HIV-infected T cells to bind to cells expressing the CD11 / CD18 receptor. In order to impair the ability of cells to bind to the CD11 / CD18 receptor, it is possible to use an antibody capable of binding to ICAM-3.

The factors of the present invention are provided to recipients in an amount sufficient to inhibit the migration of HIV / or other viral / infected T cells. The amount is considered sufficient to "suppress" the migration of T cells if the dose of the agent is sufficient to delay or prevent such migration.

The administration of such compounds can be accomplished for both therapeutic and prophylactic needs. When used prophylactically, the agents of the invention are provided in advance for any symptom of a viral infection (e.g., before, at, or immediately after the time of infection), but always before any symptom of such infection. Prophylactic administration of agents serves to prevent or delay any subsequent migration of virus-infected T cells. When used therapeutically, agents are provided at / or immediately after / the detection of virus-infected T cells. The therapeutic administration of the agent serves to delay any additional migration of such T cells.

The agents of the present invention can thus be provided both before the onset of viral infection (so as to suppress the predicted migration of infected T cells) and after the actual detection of such virus-infected cells.

D. Treatment of asthma

According to the present invention, an agent capable of modulating LFA-1 / ICAM-3 interactions is used in the treatment of asthma. In particular, the method involves administering an effective amount of an antiasthma agent to an individual in need of treatment.

The antiasthma agents of the present invention include ICAM-3, a fragment of ICAM-3, a functional derivative of ICAM-3, or anti-ICAM-3 antibodies that are capable of impairing the ability of cells to bind to LFA-1. Antibodies that bind to ICAM-3 will inhibit eosinophil migration by impairing the ability of ICAM-3 expressed on these cells to bind to cells expressing CD11 / CD18 receptors.

The anti-asthma agents of the present invention should be provided to the recipients in amounts sufficient to reduce or delay the symptoms of asthma.

The agents of the present invention may be administered either alone or in combination with one or more additional anti-asthma agents / such as methylxanthines, for example theophylline, β-adrenergic compounds, such as catecholamines, resorcinol and ephedrine, glucocorticoids, such as hydrocortisone, chromones, cathodes chromolin and anticholinergics such as atropine, with a view to reducing the amount of agents required to treat asthmatic symptoms.

The administration of the agents of the present invention can be both prophylactic and therapeutic. When used prophylactically, the agent is administered prior to any asthmatic symptom. The prophylactic administration of the agent serves to prevent or delay any subsequent asthmatic response. When used therapeutically, the agent is administered at / or immediately after / at the onset of an asthmatic symptom. The therapeutic administration of the agent serves to delay any current asthmatic episode. The agents of the present invention can thus be administered both before and after the onset of a suspected asthmatic episode.

G. Diagnostic and prognostic applications

Monoclonal antibodies capable of binding to ICAM-3 can be used as a means of suggesting or visualizing the sites of ICAM-3 expression and inflammation in a patient. In this use, anti-ICAM-3 monoclonal antibodies are labeled by radioisotopes, affinity markers (such as biotin, avidin and the like), fluorescent markers, or vapor magnetic atoms. Methods for performing such marking are well known in the art. The labeled antibodies can then be used for diagnostic assumptions. Clinical applications of antibodies in diagnostic assumptions have been described by Grossman, NV, Urol.Clin.North Amer. 13: 465-474 / 1986 /, Unger, E.C. et al., Invest.Radiol. 20: 693-700 / 1985 / and Kraw, B.A. et al., Science 209: 295-297 (1980).

ICAM-3 expression can also be detected using hybridization probes, such as mRNA, cDNA, genomic DNA, or synthetic oligonucleotide probes that bind to the ICAM-3 gene sequence, or the ICAM-3 mRNA sequences available in a cell expressing 1AM-3. Techniques for performing such hybridization tests are described by Maniatis, T.et al., In: Molec.Cloning, a Lab.Manual, Colospring Harbor, NY / 1982 /, and by Haymes.BDet al., In: Nucleic Acid Hybridization , a Practical Appr.IRL Press, Washington, DC / 1985 /.

The detection of foci of ICAM-3 expression using labeled antibodies or nucleic acid probes can be used in the diagnosis of tumors. In a diagnostic experiment, patient's tissue or blood samples were incubated in the presence of antibodies that were or could be labeled. In a preferred embodiment, this technique is carried out in a non-invasive manner by magnetic imaging, fluorography and the like. Such diagnostic tests can be used to monitor transplant recipients, such as kidneys, for early signals of potential organ rejection. Such tests may be conducted in an effort to determine the predisposition of some individuals to rheumatoid arthritis or other chronic inflammatory diseases.

For example, by radiolabeling anti-1CAM-3 antibodies or antibody fragments, it is possible to detect an antigen by radioimmunoassay. A good description of radioimmunoassay (R1A) can be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. et al., North Holland Publishing Company, NY / 1978 /, with a partial reference to the chapter entitled "Introduction to radioimmunoassay and related techniques ”by Chard, T. Conversely, the antibody may be labeled with a fluorescent compound, enzyme, or other known markers known to one skilled in the art.

In addition to localizing inflammatory sites, antibodies capable of binding ICAM-3 can be used to test biological fluids for the presence of circulating 1AM-3 using any of the commonly known fluid test media. The presence of circulating ICAM-3 in a liquid sample is indicative of an inflammatory response or other ICAM-3 mediated biological function. In addition, the presence of ICAM-3 in amniotic fluid has been associated with complications arising during pregnancy. Any biological sample can be used for a similar study, but the preferred biological fluids are: blood, serum, plasma, synovial fluids, amniotic fluids, spinal fluid or urine.

VIII. Introduction of the compounds of the present invention

The therapeutic effects of ICAM-3 can be obtained by administering to the patient the entire ICAM-3 molecule or any therapeutically active fragment thereof. Of particular importance are the therapeutically active fragments of ICAM-3, which are soluble.

ICAM-3 and its functional derivatives can be obtained synthetically, using recombinant DNA technology, by proteolysis or by a combination of similar methods. The therapeutic benefits of ICAM-3 may be enhanced by the use of functional derivatives of ICAM-3 having additional amino acid residues, for effecting attachment to the carrier or for achieving ICAM-3 activity. The scope of the present invention also extends to functional derivatives of ICAM-3, which exhibit the absence of amino acid residues or which contain modified ones as long as the derivatives or which have the biological or pharmacological activity of ICAM-3.

Both the antibodies of the present invention and the ICAM-3 molecule described herein are considered to be "substantially free from natural contaminants" if the preparations containing them are substantially pure from the materials with which these products are normally encountered. in nature.

The present invention extends to antibodies and biologically active fragments thereof (whether polyclonal or monoclonal) capable of binding ICAM-3. Such antibodies can be produced by both animals and tissue cultures, or by means of recombinant DNA technology.

The introduction of ICAM-3 or molecules derived from ICAM-3 can be performed alone or in combination with ICAM-1 or ICAM-2. The administration of anti-ICA.M-3 antibodies or other molecules capable of binding to ICAM-3 or molecules derived from ICAM-3 may be performed alone or in combination with anti-ICAM-1 antibodies and / or anti-ICAM-1 antibodies. -1CAM-2 antibodies. Administration of the patient's antibodies or fragments of antibodies capable of binding to ICAM-3, or when administered to the patient ICAM-3 (or fragments, variants or derivatives thereof), the doses of the administered agent will vary depending on such factors such as patient age, weight, gender, basic medical records, medical history, etc. In general, it is desirable to provide the recipient with a dose of the antibody that is in the range of about 1 pg / kg to 10 mg / kg (patient body weight), although it may be lower or lower. high dose. When administering ICAM-3 molecules or their functional derivatives, it is preferable to use a dose of about 1 pg / kg to 10 mg / kg (body weight), although lower or higher doses may be used . As discussed, the therapeutically effective dose may be reduced if the antiICAM-3 antibody is administered with anti-LFA-1 antibody, anti-ICAM-1 antibody, and / or anti-1CAM-2 antibody. As used herein, one compound is said to be used in addition to another when the administration of the two compounds is so close in time that they can be detected at the same time in the patient's serum.

Both the antibody capable of binding ICAM-3 and the ICAM-3 molecule itself can be administered to the patient intravenously, intramuscularly, subcutaneously, enterally, topically or parenterally. When injection of antibodies or ICAM-3 is injected, administration may be by continuous repeated injections or by single or multiple injections.

The agents of the present invention are provided to the recipient in amounts sufficient to be called "physiologically effective". The amount is called physiologically effective if the dose is sufficient to delay or prevent the physiological effect associated with ICAM-3. For example, one of the agents of the present invention is administered to a patient with the intention of suppressing inflammation and, if in a dose sufficient to do so, is said to be physiologically effective.

In addition, anti-ICAM-3 antibodies or fragments thereof may be administered either alone or in combination with one or more immunosuppressive agents (especially to an organ or tissue transplant recipient). The administration of such compounds can be both prophylactic and therapeutic.

When used prophylactically, immunosuppressive compounds are administered in advance of any inflammatory response or symptom, for example, before, at or immediately after the time of organ or tissue transplantation, but before any symptom of organ rejection. The prophylactic administration of the compounds serves to prevent or delay any subsequent inflammatory response (such as organ or tissue rejection). When administered therapeutically, the compounds are administered at / or immediately after the onset of an symptom of acute inflammation (such as organ or tissue rejection). The therapeutic administration of the compound serves to delay any current inflammation (such as organ or tissue rejection).

Thus, the anti-inflammatory agents of the present invention may be administered either before the onset of the inflammatory process (so as to delay an alleged inflammation) or after the onset of this inflammatory process.

The composition is called pharmacologically appropriate if its administration can be tolerated by the patient. This agent is administered in a therapeutically effective amount, if the amount administered is physiologically significant. An agent is physiologically significant if its presence leads to a noticeable change in the physiology of the receiving patient.

The antibodies and ICAM-3 molecules of the invention can be formulated according to known methods for the preparation of pharmacologically applicable compositions wherein these materials or their functional derivatives are combined with pharmacologically acceptable carrier vehicles. Suitable vehicles and their forms containing other human proteins, such as human serum albumin, are described, for example, in Remington Pharmaceutical Science / 16 th ed., Osol, A., Ed., Mack, Easton PA / 1980 //. For the formation of a pharmacologically suitable composition for administration to humans, such compositions will comprise an effective amount of the agent of the present invention with an appropriate amount of carrier. In addition, the antibodies of the invention may be humanized by chimerization or CDR vaccination to make them more pharmacologically relevant to the patient. Such methods are described in GB No. 9009548.0, 9009549.8 and PCT / I 91/02942 and PCT / I 91/02946.

Additional pharmaceutical methods may be used to control the duration of action. Control release agents can be carried out using polymers to absorb the agents of the present invention. The speed and duration of the control delivery can be adjusted to the required size by selecting the appropriate macromolecular matrix, varying the concentrations of incorporated molecules as well as incorporation methods. Another possible method of controlling the duration of action by control release agents is the incorporation of agents onto particles of polymeric material, such as polymers, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinyl acetate copolymers. Conversely, instead of incorporating these agents on polymeric particles, they may be enclosed in microcapsules prepared, for example, by coacervate techniques or by interfacial polymerization, by gelatin or poly / methyl methacylate / microencapsulation, or in colloidal systems, e.g. , nanoparticles and nanocapsules or macroemulsions.

The invention further encompasses pharmaceutical compositions consisting of:

a) an anti-inflammatory agent such as an antibody capable of binding ICAM-3; a fragment of this antibody capable of binding to ICAM-3; ICAN1-3, a functional derivative of ICA.M-3; or non-immunoglobulin antagonists of ICAM-3 other than ICAM-1 and ICAM-2 and in / at least one immunosuppressive agent. Examples of suitable immunosuppressive agents are: dexamethasone, azathioprine and cyclosporin A.

The invention is illustrated by the following examples, without limiting the scope thereof.

Example 1. Characterization of ICAM-3, a new adhesion ligand for LFA-1

As described above, adhesion of cell lines and lymphocytes to LFA-1 coated plates was performed / Dustin et al. Cold Spring Harbor Symp.Quant.Biol. 54: 753-765 (1989). JY-purified LFA-1 lysates were adsorbed onto 96 well microtiter plates (Linbro-titertek) at 1100 sites / pm ² . Non-specific binding sites were blocked with 1% BSA and washed with PBS (5% PBS / 2 mM MgCl2 / 0.5 HSA (assay medium)). Specific inhibition of LFA-1 is achieved by incubation for 30 min at RT microtiter wells with 1/200 dilution of TS1 / 22 / anti-LFA-1 / ascites. Resting T cells are isolated from whole blood on a plastic surface and a nylon filter and are 91% CD2 ⁺ , while PHA blasts are generated by culturing the cells in the presence of 10 pg (ml phytohaemagglutinin (PHA)). Cells were labeled with BCECF / Molecular Probes Inc./ fluorochrome, washed and resuspended in the assay medium. Mav cell treatment consists of incubation with 1/200 dilution for ascites for 45 min at 4 ° C, after which 10 ⁵ cells are transferred to each well. The cell lines adhere to the solid phase of LFA-1 for 1 h at 37 ° C, and the non-adherent cells are removed with 6 aspirations (23 gauge needle) while lymphocytes are precipitated at 30 x g for 5 min and incubated at 37 ° C in 30 min. Unbound lymphocytes which were removed by light / flick / medium from the plates 8x with 100 added between each wash. Flickering is more effective for the careful removal of unbound T lymphocytes, which are difficult to remove due to their small size. Fluorescence determination is applied directly using a fluorescence analyzer (Baxte). All MA _c are used in the diluted state (1/200 dilution of ascites) and include TS1 (22) anti-CDIA, IgCl / (Sanches-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-1C2 / 2 / anti-ICAM-1, IgC2a / de Fougerolle et al., J.Exp.Med.Submited / 1991 / I in press /, and CBR-IC3 / 1 (anti-ICAM-3, IgG1).

CBR-IC3 / 1 was obtained from a murine myeloma cell P3 X 6Aao 8.653 with splenocytes from SKW3-injected Balb / c mice / Gofter et al., Som.Cell Gen. 3: 231-236 (1977), and 600 hybridomas are screened for their ability to inhibit SKW3 binding to purified LFA-1. CBR-IC3 / 11 did not respond with purified LFA-1, nor with COS cell transfectants of ICAM-1, ICAM-2 or LFA1 cDNA (data not provided). One of the four representative experiments is shown, with the arrows showing the standard deviations.

Example 2. Cytometric analysis of cellular ICAM-1, ICAM-2 and ICAM-3 expression

All cell lines used have been described previously / Hibbs et al., J. Clin.Invest. 85: 674-681 (1990). Peripheral blood mononuclear cells are obtained by dextran sedimentation and FicollHypaque centrifugation, as described by Dustin et al., J. Cell.Biol. 107: 321-331 (1988). Neutrophils are separated from the cell pellet and contaminated erythrocytes are removed by hypotonic lysis. Lymphocytes and monocytes were separated by cytometric analysis, which was confirmed by monocytes and T-cell specific monoclonal antibodies. Adherence to a plastic material is used to enrich the PBMC lymphocytes and the cells are cultured in the presence of 10 pg / ml phytohaemagglutinin (PHA). Samples were analyzed using an EPICS Immune-Brite fluorescent beads / Coulter / for cytometer calibration. The expression of ICAM-3 on resting lymphocytes is 2-3 times greater than any CD3 or LFA-1, whereas monocytes express 3-4 times more ICAM-3 than LFA-1. The levels of ICAM-3 expression on neutrophils are equivalent to those of Mass1 / CD11 in / CD 18 /. Treatment of cells with phospholipase C showed the absence of β1-linked forms of ICAM-3.

Table 2.

Immunofluorescence cytometric assay for the correspondence between surface antigens and ICAM expression

------------------- _; ------------------------—------ = ----, Specific Intensity of N ruble Pinaeur Weevil / Tiap 'Fluorescence alCAM- 1 aICAM-2 lSAM-3 (RR1 / 1) (SWK-1C2L) (CBR-IC3 / 1) OIMPHOCYTE AT REST 1-day RNA blast 3-, tsnevii 15-day HLVejiacTH Myocyte «Eutrofmli 4 22 85 25 13 1 22 18 42 45 39 0 106 78 205 223 224 213 T lymphoblastoids SKW3 Jurkat Sup T. Molt 4 0 2 10 1 98 166 113 242 73 161 19 117 In Lycooblastoid JY SLA Ramos Raji 154 224 132 266 119 113 136 88 47 306 112 0 Monocytes U937 HL60 62 17 67 43 ! 37 203 Melanomas inc RPMI7591 896 35 475 3 0 0 ⁰ % otro lion chemical K562 293 143 0 Demolished ' HUVEC Hep G2 HCl RD3 / 5 FS1A3 A-172 31 1526 1082 0 409 0 494 41 9 0 0 0 0 0 0 ...?.

+ Membrane expression, determined by immunofluorescence cytometric analysis, is emphasized in materials and methods.

Values were determined for at least two experiments. Fluorescence droplets are used to calibrate the cytometer so that one unit is approximately 10 ³ fluorescein equivalents (Coulte Diagnostics, Hialeah, FL).

++ Mixed cell lines include: human allantoic vein endothelial cells, human mammary gland carcinoma cells, Hep C2, human epitheloid carcinoma, cell line, HeLa, human rhabdomyosarcoma RD 3/5, human fibrosarcoma and FS 1, 2, 3 , human glioblastomas.

Example 3. Immunoprecipitation of ICAM-10 3

The cell surface labeling of ¹²⁵ I was performed using an iodogen / Kishimoto et al., J. Biol. Chem. 264: 3588-3595 (1989). Cells were lysed with Triton X-100 (15 1%). The cells were clarified with bovine IgGsepharose and then incubated with appropriate MA bound _in sepharose for 2 h. The lysates were then washed and boiled in the presence of a buffer containing 50 mM Tris, 1% SDS and 1% 20 2-mercaptoethanol. The unreduced samples were boiled in buffer lacking 2-mercaptoethanol and treated with 20 mM iodoacetamide. Samples were analyzed on 7% vertical plate polyacrylamide gels as described previously by Laenmli, UKNature 227: 680685 (1970), and proteins were visualized by autoradiography. Treatment of samples with Nglicanase (Genzyme), as described previously (Tareutino et al., Biochemistry 24: 4665-4671 (1985), was carried out by concentration (10 units per ml, 37 ° C, 18 h). , gives optimal degradation of all N-linked oligosaccharides from the peptide skeleton. MHC class I contains an N-linked carbohydrate, while ICAM-2 / M _d 60, 000, 6 N-linked sites, backbone of M _g 28 383 / is a highly glycosylated.

Example 4. Distribution of ICAM-3

The anti-ICAM-3 antibodies of the present invention were used to further determine the tissue distribution of the ICAM-3 protein by cytometric analysis. Table 3 summarizes the cell types that show the surface expression of ICAM-3. It is apparent that ICAM-3 expression is restricted to hematopoietic cells. Cytometric assay data were confirmed by immunohistochemical staining of tissue areas using ICAM-3 antibodies (data not shown). As with the cytometric data, immunohistochemical studies showed that ICAM-3 expression was clearly restricted to hematopoietic cells.

Table 3.

Immunofluorescence cytometric analysis of surface antigen-related ICAM expression

Specific intensity of linear Cell Lines / Type ----- fluorescence riCAM-3 (CBR-IC3 / H a1AM-1 (RR1H) <* 1CAM-2 (CBR-IC2 / 1) • ^ pifocytes AT LEAST 4 22 - 106 1-day -PNA-shine 22 18 78 3-day J1D-b _L8ST and 85 42 205 5-day ^ ON-shiny 25 45 223 ^ onocytes 13 39 224 | "Eutrophils 1 0 ²¹³ In T-lymphoblasts were I SKW3 0 98 273 And Jurkat 2 166 161 In Sup T. 10 113 19 1 Molt 4 1 242 117

Table 3 (continued)

• In the lymphoid bla with you yes JY SLA Ramos Raji 154 224 132 266 119 113 U6 88 47 308 112 0 But you can 1 U937 62 67 37 I HL60 17 43 _ 203 Melanomas VC 896 3 0 RPMI 7591 475 0 0 Erythro leukemic K562 293 143 0 Mixed * HUVEC 31 494 0 HepG2 1526 41 0 HeLa 1082 0 0 j RD3 / 5 0 9 0 I FSL2.3 409 0 0 Ι A-172 0 0 0 1

+ Membrane expression determined by immunofluorescence cytometric analysis is as emphasized in the materials and methods. Values were determined for at least two experiments. Fluorescence droplets are used to calibrate the cytometer so that one unit is approximately 10 ³ fluorescein equivalents / Coulte Diagnostics, Hialeah, FL / ++ Mixed cell lines include: human allantoic vein endothelial cells. HUVEC, human epitheloid carcinoma cell line, human mammary gland carcinoma cells. HepG ₂ , human rhabdomyosarcoma RD 3/5 human fibrosarcoma and FS 1,2,3; human glioblastoma.

Example 5. Purification of 1AM-3

ICAM-3 was purified by immunoaffinity chromatography, whereby an anti-ICAM-3 antibody CBR-IC3 / 1 was immobilized on a matrix by known methods. Various sources have been used as starting materials for the isolation of ICAM-3. These include, but are not limited to, SKW3, the human thymus cell line and the human salivary gland. The methods used to purify ICAM-3 are mainly those described by De Fougerolles et al. in: J.Exp.Med. 175: 185-190 (1992). One of ordinary skill in the art knows that the wash and elution modes (reagents vary slightly) for each antigen and each antibody to purify them are widely used in immunoaffinity chromatography.

As noted, ICAM-3 purified from SKW3 cells or from human tonsillary cells apparently has a molecular weight of about 120 to 124 kD, while ICAM-3 purified from neutrophil lysates exhibits a molecular weight of 120 to 150 kD.

The thus purified ICAM-3 shows that they maintain the ability to bind anti-ICAM-3 antibodies, and LFA-1 to different cells / Figs. 6 and 7 /.

Example 6. Identification of different molecular weights of ICAM-3

Immunoprecipitation and polyacrylamide gel electrophoresis were used to determine the molecular weights of ICAM-3 in different cell types. The molecular weight of ICAM-3 immunoprecipitated by neutrophils was found to be different from that of lymphocyte immunoprecipitated. ICAM-3 isolated from lymphocytes and lymphoid cell lines appear as a band with a molecular weight of about 120 to 124 kD. The molecular weight of ICAM-3 isolated from neutrophils is slightly higher than that expressed by lymphoid cells, showing values of about 120 co 150 kD / fig. 4 /.

Such variations in size are not unknown to members of the ICAM family of glycoproteins. ICAM-1 demonstrates similar variations in molecular weight among different cell types. The variations in molecular weight observed for ICAM-1 are caused by variations in the extent of glycosylation. Therefore, variations in the molecular weight of ICAM-3 appear to be more determined by differences in glycosylation. Any person skilled in the art would confirm this by subjecting ICAM-3, isolated from neutrophils, to the action of various glycosidases and other enzymatic effects, and observing their effects, would see the effect on molecular weight.

Variations in the glycosylation range of ICAM-1 affect MAC-1 / CD11c / CD18 / binding. Therefore, by varying the extent of glycosylation of ICAM-3, it is possible to generate ICAM-3 derivatives having a modified affinity for binding to different ICAM-3 ligands, for example members of the CD11 / CD18 family of glycoproteins.

Example 7. ICAM-3 antibodies

Mice were injected with a combination of adjuvant and ICAM-3 protein, which was purified from KV3 or tonsil cells as described above. Monoclonal antibodies were obtained from immunized animals using procedures known in the art.

Table 4 summarizes the variants of anti-ICAM-3 antibodies. The various antibodies have been identified based on their ability to react against purified immobilized ICAM-3. Positive MAs _are then screened on various cell lines known to be ICAM-3 positive, and then immunoprecipitated by radiolabeled ICAM-3 positive cell lysates. MAs were then tested for their ability to block PMA-induced SKW3 cell aggregation in the presence of anti-ICAM-1 and anti-1CAM-2 antibodies. Conversely, the anti-ICA.M-3 antibody can be identified by its ability to inhibit the binding of SK.W3 cells to immobilized purified ICAM-3.

One of the lines proving that the third ligand of LFA-1 exists is the presence of ICAM-1 / ICAM-22 independent way for PMA stimulated SKW3 cell aggregation. We were able to block this aggregation with both a murine anti-1CAM-3 polyclonal antiserum and a combination of CBR-IC3 / 1 and CBR-IC3 / 2.

Table 4.

ICAM-3 mAbs

mAb Isotype ΡΜή. -indupirrnn Aggregation · In-house telnc + IC1 + 2 mAb + IC1 + 2 + IC3 / 1 mAb CBR-IC3 / 2 IgG2a, k 3 2 0 CBR-IC3 / 3 IgGZa, k 4 3 2 CBR-IC3 / 4 IgM, k 4 3 2-3 CBR-JC3 / 5 IgG2a, k 2 2 The scoreboard is now 0-1 CBR-IC3 / 6 N.D. 5 5 5 1CAM-3 antisera 3 0 0 CBR-IC3 / 1 IgGl, k 5 4 - HP2D9 ·· 1

+ Refers to the scale of J.Exp.Med. 1991 paper = aggregation = aggregation number ** Another ICAM-3 mAv from headquarters in Spain - Sanchez-Madrid.

After presenting our results, they remembered the mAv they received, but never characterized. They consider it ICAM-3

CBR-IC3 / 2, 3/3, 3/5 immunoprecipitated the 120 kD band as CBR-IC3 / 1.

Example 8. Immunological studies with anti-ICAM-3 antibodies

Anti-ICAM-1 antibody / RR1 / 1 /, anti-ICAM-2 antibody / CBR-IC2 / 2 /, and anti-ICAM-3 antibodies / combination of CBR-IC3 / 1 and CBR1C3 / 2 / are tested for their ability to block (1) PMA-stimulated SKW3 cell adhesion against purified ICAM-1; (2) phytohaemagglutinin (PHA) stimulation of cell division, and (3) response of mixed lymphocytes.

Previously, it has been shown that PMA can activate LFA-1, thereby activating their ability to bind to ICAM-1. / Dustin et al., Nature 341: 619 (1989). Figure 5 shows that similar activation mechanisms are present in LFA-1 / ICAM-3 binding.

The ability of different antibodies to block the binding of PMA stimulated KV3 cells to purified ICAM-3 and ICAM-1 has been investigated.

Figure 6. The CBR-1C3 / 1 and CBRIC3 / 2 antibodies, when present alone, did not effectively block the binding of PMA stimulated SKW3 cells to purified ICAM-3. At the same time, PMA-stimulated SKW3 cellular binding to ICAM-3 can be blocked by a combination of these two antibodies. As noted by De Fougeroll in J.Exp.Med., The CBR-IC / 3/1 monoclonal antibody is itself capable of blocking the binding of ICAM-3 to purified LFA-1.

The binding of PMA-stimulated SKW3 cells to ICAM-3 was further analyzed to determine the likelihood of temperature and cation dependence. As with ICAM-1, ICAM-3 / LFA-1 binding has been found to be both temperature-dependent and cation-dependent. FIG. 8 / / data not shown /.

Figure 9 summarizes the effect of different antibodies on PHA stimulation of cell division. As noted earlier / Krensky et al., J. Immunol. 131: 611-616 (1983), PHA stimulates cell proliferation such that it is inhibited by anti-LFA-1 in anti-1CAM-1 antibodies, anti1CAM-2 antibodies, or a combination of antiICAM-1 and anti-1CAM-2 antibodies, so it has little effect on PHA stimulated cell division. At the same time, a combination of: 1 / anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3 antibodies;

2 / two anti-ICAM-3 antibodies (ICC / 1 and ICC / 2)

3 / anti-ICAM-1 and anti-ICAM-3 antibodies are effective at blocking PHA stimulated cell division.

Example 9. Response of mixed lymphocytes

The mixed lymphocyte response assay (MLR) was used to assess immunological reactivity. The test involves the basic addition of chemically fixed foreign cells (stimulatory cells) to a solution containing PBLs from different individuals (responding cells) and measuring the response level using the proliferation of responding cells as an index / described previously by Krensky et al., J. Immunol. 131: 611-616 / 1983 //. This test is the first step in identifying the composition applicable to the treatment of tissue transplant rejection.

The effect of different antibodies and combinations of antibodies on MLR reactions is presented in FIG. 10. In general, antiICAM-3 alone shows a low impact level. However, a higher level of M1P blocking efficiency was found to occur with a combination of anti-ICAM-1 and anti-ICAM-3 antibodies. The highest result was obtained with a combination of anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3 antibodies.

Example 10. Peptide sequences of ICAM-3

ICAM-3 was purified from human tonsillar cells by immunoaffinity chromatography as described in Example 5. Purified ICAM-3 was enzymatically digested with the lys-C enzyme in a known manner and the peptide fragments were dissolved by HPLC. FIG. 11 shows the gas chromatography of the observed peaks of different proteins upon typical degradation. Peaks 10 and 17 were identified as containing peptide fragments of satisfactory size and structure for alignment (hereinafter NK-10 and NK-17, respectively).

The amino acid sequence of NK-17 and NK-10 peptides was determined by standard procedures. The NK-10 peptide sequence, as found, is KIDRATCPQHIK /SEQ.JD.NO.1/. The first lysine (K) residue included in the sequence is considered to be present insofar as Lys-C breaks down proteins after the lysine residue.

The NK-17 peptide sequence was found to be KIALETSISK /SEQ.ID.NO.2/. As with NK-10 protein, the first lysine residue was assumed and later confirmed by DNA sequencing analysis.

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

Example 11. cDNA cloning of ICAM-3

On the basis of the peptide sequences obtained above, degenerate oligonucleotide probes are produced. The latter are further engineered to include a previously identified codon observed within the ICAM-1 and ICAM-2 nucleotide sequences. The nucleotide sequences of the probe based on those of NK-10 and NK-17 proteins are as follows:

5'-AAN-GAN-GTC-TCC-AGG-GCTATC-TT-37SEQ.NO.3 /

NK-17 probe. Oligo 23 measure with 24x degeneration containing 2Ns (inosine).

5'-TNN-AGA-TGT-TGN-GGG-CANGTN-GCN-C 3 / SEQ.lD.No 4

NK-10 probe. 25 8-fold measure containing 5Ns (inosine).

The cDNA expression library was obtained from tonsillar RNA, as described previously / Wong et al., Proc.Natl.Acad.Sci. USA 82: 7711-7716 / 1985 /. The library is screened using a NU-17 probe and plaques showing positive hybridization are screened with the NK-10 probe. One branch, branch 11.2, is found to have an insert of about

I, 6 to 1.8 sq. In length. The diagrammatic representation of clone 11.2 and localization of NK-10 and NK-17 peptides is shown in FIG. 12.

The ends of the inserts are coordinated using techniques known to the person skilled in the art, using primers obtained from the articulating vector, while the NK-10 probe is used as an internal sequencing primer. The sequences thus presented are read in only one chain sequence. Any person skilled in the art would appreciate that such a sequence can be made to work in routine ways.

The common sequences obtained are presented in FIG. 18. There are several things to note in this order. First, a sequence with a high degree of homology to the transmembrane region of ICAM-1. Therefore, one who wishes to produce soluble fragments of ICAM-3 should deletion all sequences after the onset of the transmembrane region indicated in FIG. 12. Second, the sequence obtained from the 5-end of the clone

II, 2 appears to be close to the 5-terminus of the naturally occurring agent, as may be suggested by the sequential homology that exists between ICAM-1, ICAM-2 and ICAM-3.

Example 12. Sequence homology between different ICAM molecules

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

The results show that ICAM-3 shows a high level of homology with both ICAM-1 and ICAM-2. Sequence homology between ICAM-1 and ICAM-3 was established within the first (Fig. 16) and fourth / fifth regions of ICAM-1, as well as the transmembrane region and some of the cytoplasmic region of ICAM-1 (Fig. 13 and 14). Further studies on gene bank data indicate the absence of other sequences identical or similar to those obtained for 1AM-3. Significant homology was also observed between ICAM-3 and the first ICAM-2 regions (Fig. 17).

Branch 11.2 was used to further screen libraries. Other clones have been found to have cDNA inserts of about 2 to 2.4 kD. As if this represents cDNA clones along their entire length, as long as nothing larger is identified.

Claims (52)

1. ICAM-3 or its functional derivatives, substantially free from natural contaminants. 2. A recombinant DNA molecule capable of encoding ICAM-3 or its functional derivatives. The recombinant DNA molecule according to claim 2, which is additionally capable of expressing ICAM-3 or its functional derivatives in a cell. An antibody or antibody fragment capable of binding to a molecule selected from the group consisting of ICAM-3 and its functional derivatives. The antibody of claim 4, wherein the binding of the antibody to said molecule impairs the ability of the same molecule to bind to a receptor molecule. The antibody of claim 5, wherein said receptor is LFA-1. The antibody of claim 5, wherein said receptor is MAC-1. The antibody of claim 5, wherein said receptor is p150.95. A hybridoma cell capable of producing the monoclonal antibody of claim 4. A method for producing a desired hybridoma cell to produce an antibody capable of binding to ICAM-3, or bifunctional derivatives thereof, comprising the following steps: - immunization of an animal with purified 1AM-3; - fusion of splenocytes from the animal with a myeloma cell line; c) allowing the fusion of splenocytes and myeloma cells to form a hybridoma cell secreting an antibody; and d / screening the hybridoma cells for the desired hybridoma cell capable of producing an antibody to bind to ICAM-3. 11. A method for modulating cell biological functions mediated by ICAM-3, comprising providing the person in need of treatment with an effective amount of ICAM-3 modulating agent, which in turn is selected from the group consisting of : an antibody capable of binding to ICAM-3; a fragment of this antibody that is also capable of binding to ICAM-3; ICAM-3; its functional derivatives; and non-immunoglobulin antagonists of ICAM-3 other than ICAM-1, ICAM-2 or members of the CD-18 molecular family. The method of claim 11, wherein said ICAM-3 mediated biological functions are an inflammatory response and said ICAM-3 modulating agent is provided to the subject in an amount sufficient to suppress the inflammation. The method of claim 12, wherein said inflammatory response is specific inflammation in response to a condition selected from the group comprising: delayed type hypersensitivity reaction, psoriasis symptom, autoimmune disease, organ or tissue rejection after transplantation. The method of claim 13, wherein said organ is a solid organ transplant. The method of claim 14, wherein the organ transplant is a kidney. The method of claim 13, wherein the organ transplant is a soft tissue transplant. The method of claim 16, wherein the transplant is a bone marrow. The method of claim 13, wherein said autoimmune disease is selected from the group consisting of: Reynaud syndrome, autoimmune thyroidase, EAE, multiple sclerosis, rheumatoid arthritis and lupus erythematosus. The method of claim 13, wherein the inflammatory response is nonspecific inflammation in response to conditions selected by a group consisting of: ARDS / multiple organ damage secondary to septicemia, trauma or hemorrhage, reperfusion damage to myocardial or other tissues, acute glomerulonephrosis, reactive arthritis, dermatitis with acute inflammatory components, acute purulent meningitis or other disorders of the central nervous system due to inflammation (eg stroke), thermal but damage, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis associated with granulocyte transfusion syndromes and cytokine-induced toxicity. The method of claim 11, wherein the ICAM-3 mediated functions are metastases of hematopoietic tumor cells, these cells requiring a functional member of the CD-18 molecular family to migrate and said agent provided to the subject in an amount sufficient to suppress said metastases . The method of claim 11, wherein the ICAM-3 mediated biological functions are extracellular migration of virus-infected leukocytes and said ICAM-3 modulating agent is introduced into the subject having such leukocytes in an amount sufficient to suppress their migration. The method of claim 21, wherein said virus-infected cells are infected with HIV. 23. The method of claim 11, wherein said ICAM-3 biological function is cell migration associated with an asthmatic response, and said ICAM-3 modulating agent is provided to the subject in an amount sufficient to suppress cell migration associated with asthma . 24. A method of suppressing HIV infection of a leukocyte, which method comprises administering to a subject exposed to or infected with HIV an effective amount of an HIV-1 infectious suppressant agent selected from the group comprising: an antibody capable of bind to ICAM-3, a toxin derivative of this antibody; an antibody fragment capable of binding to ICAM-3, a toxin derivative of that fragment; ICAM-3; 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 molecular family; a toxin derivative of this member of the CD-18 molecular family. The method of claim 24, wherein said HIV is HIV-1. 26. A method of inhibiting the development of an ICAM-3 expressing tumor cell, which comprises providing an amount of toxin sufficient to inhibit development, this toxin being selected from the group consisting of: an antibody capable of binding to ICAM-3; a toxin derivative of this antibody; a fragment of this antibody, which fragment is capable of binding to ICAM-3; a toxin derivative of this fragment; a toxin derivative of a member of the CD-18 molecular family and a toxin derivative functionally of a member of the CD-18 molecular family. 27. A method of inhibiting the growth of LFA1-expressing tumor cells, which comprises providing a toxin sufficient to suppress growth, this toxin being selected from the group consisting of toxin derivatives of ICAM-3 and toxin derivatives functionally of ICAM- 3. The method of any one of claims 11 to 27, wherein the method further comprises introducing one or more agents selected from the group consisting of: an antibody capable of binding to LFA-1, a fragment of that antibody , as this fragment is capable of binding to LFA-1, a non-immunoglobulin antagonist of LFA-1, an antibody capable of binding to ICAM-1, a fragment of this antibody, being capable of binding to ICAM-1, an antibody capable of binding to ICAM-2, a fragment of that antibody, being capable of binding to ICAM-2 . The method of any of claims 11 to 27, wherein the ICAM-3 modulating agent is administered enterally, parenterally, topically, as an inhaler or intranasally. The method of any one of claims 11 to 27, wherein the ICAM-3 modulating agent is prophylactically administered. The method of any of claims 11 to 27, wherein the ICAM-3 modulating agent is administered therapeutically. The method of claim 29, wherein the parenteral administration is performed by intramuscular, intravenous or subcutaneous means. 33. A pharmaceutical composition comprising an agent selected from the group consisting of: an antibody capable of binding to ICAM-3, a fragment of that antibody, this fragment being capable of binding to ICAM-3, ICAM-3, FIG. a functional derivative of ICAM-3 and a non-immunoglobulin antagonist of ICAM-3 other than ICAM-1, ICAM-2 or a member of the CD-18 family. The pharmaceutical composition of claim 33, further comprising an immunosuppressive agent. A pharmaceutical composition comprising the ICAM-3 modulating agent of any of claims 11, 24, 26, 27 or 28, in combination with a pharmacologically suitable carrier. The pharmaceutical composition of claim 35, in combination with at least one other immunosuppressive agent. 37. A therapeutic composition comprising an antibody molecule or fragment thereof according to claim 4, in combination with a pharmacologically suitable solvent, excipients or carriers. A diagnostic composition comprising an antibody molecule or fragment thereof according to claim 4 in a detectable form. 39. A method for detecting the presence of cells expressing ICAM-3, which includes: a) incubation of said cells or cell extract in the presence of a nucleic acid molecule, which molecule is capable of hybridizing with ICAM-3 mRNA and c) determining whether said nucleic acid molecule has hybridized to a complementary nucleic acid molecule present in the given nucleic acid molecule cell or extract of these cells. 40. A method for diagnosing mammalian ICAM3-expressing tumor cells, comprising: a) introducing to the subject a composition comprising a detectable antibody or a fragment thereof capable of binding to ICAM-3 and c) detecting the binding of said antibody to said ICAM-3. 41. A method for diagnosing mammalian inflammation, comprising: a) incubating a sample comprising the tissue of the subject with a composition comprising an antibody or fragment thereof capable of binding to a cell expressing 1AM-3 and c) detecting the binding of said antibody to said cell. 42. A method for diagnosing the presence of 1CAM-3 in a liquid sample taken from a mammal that includes: a) incubating the sample with a biological fluid taken from said subject with a composition comprising an antibody or fragment thereof capable of binding to ICAM-3 and c) detecting the binding of said antibody to said liquid. The method of claim 42, wherein said fluid is selected from the group consisting of: blood, serum, plasma, synovial fluid, amniotic fluid or urine. The functional derivative of claim 1, wherein the derivative is a fragment of ICAM-3. The fragment of claim 44, which is selected from the group consisting of: Seq.ID Nos. 2, 4 and 6. The functional derivative of claim 2, wherein the derivative is a fragment of said DNA molecule capable of encoding ICAM-3. The fragment of claim 45, which is selected from the group consisting of Seq.ID Nos. 1, 3, and 5. The fragment of claim 44, which is selected from the group consisting of IC1-D1, D1-2, D1-3, D1-4, and D1-5. 49. A method for identifying agents capable of modulating ICAM-3 binding to LFA-1, comprising the step of contacting ICAM-3 with LFA-1 in the presence of said agent and measuring the modulation of ICAM-3 / LFA-1 binding . The method of claim 49, wherein said LFA-1 and said ICAM-3 are purified with a view to removing naturally occurring contaminants. The method of claim 49, wherein said LFA-1 is expressed on the cell surface and said ICAM-3 is purified from naturally occurring contaminants. The method of claim 49, wherein said ICAM-3 is expressed on the cell surface and said LFA-1 is purified from naturally occurring contaminants.