JP3288042B2 - Intercellular adhesion molecule-3 and its binding ligand - Google Patents

Description

Description: BACKGROUND OF THE INVENTION Cross-Reference of Related Applications This application is a subject of U.S. patent application Ser. No. 07 / 712,879 (June 1991).
No. 07 / 045,963 (filed on May 4, 1987) and US patent application Ser.
No. 98 (filed on November 2, 1987), No. 07 / 155,943 (filed on February 16, 1988), No. 07 / 189,815 (filed on May 3, 1988), and No. 07 / 250,446 (1988 Filed on September 28), No. 07/4
No. 54,294 (filed on December 22, 1989), PCT Application No. PCT / US91 /
No. 02942 and PCT / US91 / 02946 (filed with the US Receiving Office on April 27, 1991). These applications are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION The present invention relates to the discovery of a novel intercellular adhesion molecule, termed ICAM-3. This molecule is involved in the process when a population of leukocytes recognizes and attaches to cellular substrates. ICAM-3 mediates the interaction of cells with other lymphocytes, macrophages, and neutrophils at the site of inflammation and the site of the immune response.

The invention further relates to using ICAM-3 alone or in combination with ICAM-1 and / or ICAM-2 to inhibit cell-cell adhesion of granulocyte, lymphocyte, or macrophage lineages. The use of such molecules provides a treatment for specific and non-specific inflammation.

The present invention further provides for the administration of ICAM-3 alone or in combination with ICAM-1 and / or ICAM-2 to achieve leukocyte HIV infection, particularly HIV infection, in individuals infected or exposed to HIV. -1 relates to a therapeutic method and a preventive method for suppressing infection. Therefore, the present invention relates to HIV
Provide therapy for diseases such as AIDS (acquired immunodeficiency syndrome) caused by the virus.

The present invention further relates to a therapeutic method for inhibiting the migration of HIV-1-infected cells from the circulatory system using ICAM-3 alone or in combination with ICAM-1 and / or ICAM-2. Thus, the present invention provides a therapy for diseases such as AIDS (acquired immunodeficiency syndrome) 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 to kill T cells and "syncytia" (syncytia) in HIV-infected individuals.
ia) Therapeutic methods that suppress formation. Therefore, the present invention
It provides a therapy for diseases such as AIDS (acquired immunodeficiency syndrome) caused by the HIV-1 virus.

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

The present invention further relates to a molecule capable of binding to ICAM-3 (hereinafter, referred to as ICAM-3).
Anti-ICAM-3). Anti-ICAM-3 molecule ICAM-
Binding to 3 is intended to modulate a biological function for ICAM-3. A binding molecule of the invention can be an antibody, peptide, or carbohydrate capable of binding ICAM-3. Such binding molecules are useful for modulating the biological function 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 to inhibit cell-cell adhesion of granulocyte, lymphocyte, or macrophage lines. The use of such molecules provides a method of treating specific and non-specific inflammation.

The present invention also provides for leukocyte HIV infection in individuals infected or exposed to HIV by administering anti-ICAM-3 alone or in combination with anti-ICAM-1 and / or anti-ICAM-2. In particular, it relates to a therapeutic method and a preventive method for suppressing HIV-1 infection. Therefore, the present invention
Provide therapy for diseases such as AIDS (acquired immunodeficiency syndrome) caused by the HIV virus.

The present invention further provides an anti-ICAM-3 agent alone or an anti-ICAM-3 agent.
The present invention relates to a therapeutic method for suppressing the migration of HIV-1 infected cells from the circulatory system using one agent and / or a combination with an anti-ICAM-2 agent. Thus, the present invention provides a therapy for diseases such as AIDS (acquired immunodeficiency syndrome) caused by the HIV-1 virus.

The present invention further provides an anti-ICAM-3 agent alone or an anti-ICAM-
It relates to the use of one agent and / or a combination with an anti-ICAM-2 agent in the treatment of asthma.

Description of the Related Art A. Leukocyte Attachment and Function To properly protect the host from foreign invaders such as bacteria or viruses, leukocytes must be able to attach to the cell substrate. These functions are described in Eisen, H.
W., (In: Microbiology, Third Edition, Harper & Row, Philade
lphia, PA (1980), pp. 290-295 and 381-418). Leukocytes must be able to attach to endothelial cells so that they can migrate from the circulatory system to the site of inflammation. further,
Leukocytes are used so that a normal specific immune response can occur.
It must attach to antigen presenting cells and, finally, to the appropriate target cells so that lysis of virus-infected cells or tumor cells occurs.

Recently, leukocyte surface molecules involved in mediating the adhesion-mediated functions described above have been identified using hybridoma technology. Briefly, a monoclonal antibody against human T cells that binds to leukocyte surfaces and inhibits the aforementioned adhesion-related functions (Davignon, D et al., Proc. Natl. Acad. Sci. USA 7
8: 4535-4539 (1981)) and monoclonal antibodies against mouse spleen cells (Springer, T. et al., Eur. J. Immunol.
9: 301-306 (1979)) (Springer, T. Fed. P.
roc. 44: 2660-2663 (1985)). Molecules identified by these antibodies were Mac-1 and leukocyte function-related antigens.
1 (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found on lymphocytes (Springer, TA et al., Immunol. Rev. 68:
111-135 (1982)). These two molecules, as well as a third molecule, p150,95, which has a similar tissue distribution to Mac-1, play a role in cell adhesion (Keiz
er, G. et al., Eur. J. Immunol. 15: 1142-1147 (1985)).

The leukocyte molecule described above was found to be a member of a related family of glycoproteins, called the "CD-18 family" of glycoproteins (Sanchez-Madrid.
FJ et al., Exper. Med. 158: 1785-1803 (1983); Keizer, G.
D. et al., Eur. J. Immunol. 15: 1142-1147 (1985)). This glycoprotein family consists of one alpha chain and one
It consists of a heterodimer with two beta chains. The alpha chains of the antigen are different, but the beta chains are
Highly preserved (Sanchez-Madrid, FJExper, Me
d.158: 1785-1803 (1983)). The beta chain of the glycoprotein family (sometimes called “CD18”) is 95 kd
Is found to have a molecular weight of
It turned out to vary from 150kd to 180kd (Spring
er, T., Fed. Proc. 44: 2660-2663 (1985)). Although the alpha subunit of the glycoprotein does not show as much homology as the beta subunit, a close analysis of the alpha subunit of the glycoprotein showed that they were substantially similar. For similarities between the alpha and beta subunits of LFA-1-related glycoproteins, see Sanchez-Madrid, F. et al. (J. Ex.
per.Med. 158: 586-602 (1983)); J.Exper.Med. 158: 178.
5-1803 (1983)).

A group of individuals that cannot express normal amounts of any of these adhesion protein families on leukocyte cell surfaces has been identified (Anderson, DC et al., Fed. Proc. 44: 2671-2).
677 (1985); Anderson, DC et al., J. Infect. Dis. 152: 668.
−689 (1985)). Such individuals are known as "defective leukocyte adhesion" (LAD) (Anderson, DC et al., Fed. Proc. 44: 2671).
−2677 (1985); Anderson, DC et al., J. Infect. Dis. 152−
668-689 (1985)). Characteristics of LAD patients include necrotic soft tissue lesions, impaired pus formation, and wound healing, as well as abnormal adhesion-dependent leukocyte function in vitro and susceptibility to chronic and recurrent bacterial infections. Granulocytes from LAD patients behave in an incomplete manner in vitro, much as normal granulocytes do in the presence of anti-CD18 monoclonal antibodies. That is, these granulocytes cannot exert adhesion-related functions such as aggregation or adhesion to endothelial cells. More importantly, however, it was observed that such patients were unable to enhance a normal inflammatory response because their granulocytes were unable to adhere to the cellular matrix.
Most notably, these LAD-derived granulocytes cannot adhere to endothelial cells in blood vessels near inflammatory lesions,
It was observed that sites of inflammation such as skin inflammation could not be reached. Such attachment is a necessary step for extravasation.

Lymphocytes from these patients showed similar defects in vitro as normal lymphocytes, where molecules of the CD18 family were neutralized by antibodies. Furthermore, such individuals failed to enhance the normal inflammatory response due to the inability of the cells to adhere to the cellular matrix (An
derson, DC et al., Fed. Proc. 44: 2671-2677 (1985); Ande.
rson, DC et al., J. Infect. Dis. 152: 668-689 (1985)).
These data indicate that the immune response is attenuated if lymphocytes cannot adhere in the normal way due to a deficiency in the functional adhesion molecule of the CD18 family.

Thus, in summary, maintaining the health and life of an animal requires the ability to adhere to other cells of leukocytes, such as endothelial cells. It has been found that this adhesion requires cell-cell contact involving specific receptor molecules present on the cell surface of leukocytes. These receptors allow leukocytes to adhere to other leukocytes, endothelial cells, and non-vascular cells. Cell surface receptor molecules LFA-1, Mac-1, and p15
0,95 have been found to be closely related to each other. In humans with leukocytes lacking these cell surface receptor molecules, chronic and recurrent infections, as well as other clinical symptoms, including incomplete antibody responses, appear.

Furthermore, since leukocyte adhesion is involved in the process by which foreign tissue is identified and rejected, understanding of this process is based on transplantation of individual organs such as the kidney, transplantation of non-solid organs such as the bone marrow, tissue transplantation, allergy, oncology There is significant value in the field.

B. HIV Infection HIV infection is a cause of AIDS. Many HIV variants have been reported. The main ones are HIV-1 and HIV
-2. HIV-1 is endemic in North America and Europe, and HIV-2 is only prevalent in Africa. These viruses have similar structures and encode proteins with similar functions. HIV infection involves a viral protein (called "gp120")
4 ("T helper") is thought to occur by binding to a receptor molecule (referred to as "CD4") present on the surface of lymphocytes (Schnittman, SM et al., Incorporated herein by reference). , J. Immunol. 141: 4181
-4186 (1988)). After binding to this receptor, the virus enters the cell, replicates, and kills T cells in the process. Thus, the destruction of an individual's T4 cells is a direct consequence of HIV infection.

T cell destruction compromises infected patients' ability to fight opportunistic infections. Cancer often occurs in individuals with AIDS, but the relationship between such cancer and HIV infection is largely unknown.

Although slight replication of the HIV virus is also necrotic to infected cells, such replication is typically detected in only a small portion of T4 cells in infected individuals. Recent results indicate that more viremia occurs than previously thought, and that T cell infection rates are as low as 1%.

Several studies have elucidated other mechanisms by which the HIV virus mediates T4 cell destruction.

Apart from HIV replication, HIV infected cells can be destroyed by the action of cytotoxic killer cells. Killer cells are normally present in humans and serve to monitor the host and destroy any foreign cells it encounters (eg, found in incompatible blood transfusions or organ transplants). When infected with HIV, T4 cells display gp120 molecules on their cell surface. Killer cells recognize such T4 cells as foreign (rather than as native cells) and therefore mediate their destruction.

HIV infection can also lead to the destruction of healthy cells that are not infected. Infected cells can secrete the gp120 protein into the bloodstream. The released gp120 molecules can then bind to the CD4 receptor on healthy uninfected cells. With such binding, the cells assume the appearance of HIV-infected cells. Cytotoxic killer cells recognize gp120 bound to uninfected T4 cells, recognize the cells as foreign, and mediate the destruction of the cells.

Yet another mechanism, of particular interest to the present invention, is the mechanism by which HIV causes T4 cell killing by "synchthia" formation. "Synchium"
It is a multinucleated giant cell, formed by the fusion of hundreds of T4 cells. HIV infection allows infected cells to have the ability to fuse with other, infected or non-infected healthy T4 cells. Syncytium cannot function and die quickly. The killing results in the destruction of both HIV infected and non-HIV infected T cells. This process is of particular interest for the present invention as it requires direct cell-cell contact of T4 cells. The ability of HIV-infected cells to form syncytia suggests that such cells have gained a means to fuse with healthy cells.

HIV infection, and particularly HIV-1 infection, appears to affect cell surface expression of leukocyte integrins and cell adhesion responses mediated by these heterodimers (Petit, AJ et al., J. Clin. Invest. 79: 188
(1987); Hildreth, JEK et al., Science 244: 1075 (198
9); Valentin, A. et al., J. Immunology 144: 934-937 (199
0); Ressen, RD et al., Trans.Assoc.American Physicians
102: 117-130 (1990), all of which are incorporated herein by reference). After HIV-1 infection, U93
Increased homotypic aggregation of 7 cells, as well as cell surface expression of CD18 and CD11b (Petit, AJ et al., J. Clin. Invest. 79: 1
88 (1987)). U937 cells infected with HIV-1 are uninfected U937 cells.
Much easier to adhere to IL-1-stimulated endothelial cells than 937 cells.
This behavior can be suppressed by treating the endothelial substrate with an anti-CD18 or anti-CD11a monoclonal antibody or with an anti-ICAM-1 antibody (Rossen, RD, Trans. Assoc. American Physicians 102:
117-130 (1989)). Monoclonal antibodies against CD18 or CD11a also include phytohemagglutinin (PHA)
Stimulated lymphoblast cells and constitutively infectious CD
4 It has been shown that it can inhibit the formation of syncytia, including negative T cells (Hildreth, JEK et al., Science 2
44: 1075 (1989)). Treatment of virus-infected cells only with anti-CD18 or anti-CD11a monoclonal antibodies showed little effect on syncytia formation. This suggests that these antibodies primarily protect uninfected target cells from infection (Hi
ldreth, JEK et al., Science 244: 1075 (1989); Valenti
n, A. et al., J. Immunology 144: 934-937 (1990)). Valen
tin et al. (Valentin, A. et al., J. Immunology 144: 934-937 (1
990)) recently demonstrated that monoclonal antibodies specific for CD18 inhibit syncytia formed when continuous T cell lines were cultured with HIV-1 infected U937 cells. It was confirmed.

The mechanism by which a monoclonal antibody specific for CD18 or CD11a prevents susceptible cells from fusing with HIV-infected cells remains unclear and is not required in the present invention, but was studied in radiolabeled gp120 studies. CD18
It has been suggested that heterodimers, including, do not provide a site for binding to the virus (Valentin,
A. et al., J. Immunology 144: 934-937 (1990)). Thus, HIV infection includes cell-cell interactions and / or virus-cell interactions that mimic such cell-cell interactions. This cell-cell interaction can result in transport of cell-free virus across the endothelial barrier or transport of virus-infected cells. Virus-cell interactions similar to cell-cell interactions may facilitate or enable free virus to attach and / or infect healthy cells.

Thus, the present invention relates, in part, to HIV infection and, in particular, HIV infection.
-1 infection comes from the observation that CD11a / CD18 heterodimer expression and its binding ligand expression are increased. This increased expression is important in that it increases the ability of HIV-infected T cells to attach to and aggregate with one another (ie, “homogeneous aggregation” occurs). This finding suggests that this type of agglutination does not occur between resting normal leukocytes, and this finding suggests that CD11 / C
It is suggested that ligands such as the D18 receptor and / or ICAM-1 are required for such aggregation.
For homotypic aggregation to occur, LFA-1 must bind to ICAM-1. As described herein, IC
AM-3 is the only ICAM family molecule that is expressed at high levels on resting T cells. Unless the T cells are "activated", only the anti-ICAM-3 antibody will
Can be blocked from bonding to Thus, anti-ICAM-3 antibodies can be used to suppress T cell aggregation.

In addition, anti-ICAM-3 antibodies can be used to inhibit the adhesion process of infected T cells. This adhesion process assists HIV-1 in migrating from infected cells to healthy cells in an individual and helps or promotes healthy cells to become infected with free virus.

C. Migration of HIV-Infected Cells Migration and scattering of leukocytes is important in protecting individuals from infection. However, these processes also play a role in the migration and scatter of leukocytes infected with the virus. Of particular importance is the migration and scatter of leukocytes infected with HIV. With such cell migration,
The formation of extravascular foci is made and can cause tumors and other abnormalities.

Histological examination of affected organs reveals extravascular mononuclear cell infiltration of the lesion. Attempts to identify cells infected with the virus in such infiltrates in the central nervous system have revealed the presence of HIV-1 infected cells. These studies have shown that the HIV-1 virus is primarily present on monocytes and macrophages, and other cells of this lineage (RT Johnson et al., FASEB
J. 2: 2970 (1988); MHStoler et al., J. Amer. Med. Assn. 25
6: 2360 (1986); S. Gartner et al., Science 233: 215 (198
6)).

The mechanism that stimulates the formation of extravascular infiltrates of HIV-1 infected monocyte-like cells has heretofore been poorly understood. This mechanism may involve either the transport of virus released from cells or the transport of virus through the endothelial barrier in the cytoplasm of infected mononuclear cells.

HIV-1 infection stimulates the expression of cell surface molecules that promote leukocyte adhesion to vascular endothelial cells and migration of leukocytes from the blood to extravascular tissue sites (CWSmith
J. Clin. Invest. 82: 1746 (1988), which is incorporated herein by reference). Therefore, it has been proposed to prevent the spread of HIV-infected cells by using an antibody that inhibits cell migration (WO 90/13316).

D. Asthma: Clinical Features Asthma is a heterogeneous family of diseases. Asthma is characterized by a tracheobronchial hyperreactivity to stimuli (Kay, AB, Allergy and Inflammation, Academic Pres
s, NY (1987); which is incorporated herein by reference). Clinically, asthma manifests as tracheobronchial stenosis, dense obstructive secretions, and attacks of dyspnea, cough, and Zeyze. The relevance of each of these symptoms is unknown, but the end result is increased airway resistance, hyperinflation of the lungs and thorax, and abnormal ventilation distribution and abnormal blood flow in the lungs. In this disease, acute symptoms appear for a temporary period during a period of no symptoms. Acute symptoms can be hypocarbonic and fatal. About 3% of the world's population suffers from asthma.

Allergic asthma and self-mixing (idiosyncratic)
Two types of asthma have been reported. Allergic asthma is usually associated with hereditary allergic diseases such as rhinitis, gin machine and eczema. Symptoms include positive wheal and wheal-and-flar for intradermal injection of airborne antigens (eg, pollen, environmental contaminants, occupational contaminants, etc.).
e) Characterized by response, as well as increased serum levels of IgE. Allergic asthma appears to be causally related to the presence of IgE antibodies in many patients. Asthmatics that do not display the above characteristics are considered idiosyncratic asthma.

Allergic asthma relies on an IgE response controlled by T and B lymphocytes and is thought to be activated by the interaction of air-borne antigens with preformed IgE molecules bound to mast cells Have been. When encountering an antigen, the antigen is used for a long period to sensitize the individual.
It must be at a concentration sufficient to produce IgE. Once sensitized, asthmatics become responsive in response to very low levels of antigen.

Asthma symptoms can be exacerbated by the presence and increase of triggering antigens, environmental factors, occupational factors, physical work, and emotional stress.

Asthma includes methylxanthine (such as theophylline), beta-adrenergic antagonists (such as catecholamines, resorcinol, saligenin, and ephedrine), glucocorticoids (such as hydrocortisone), and inhibitors of mast cell degranulation (ie, such as cromolyn sodium). Chromone), and anticholinergics (such as atropine).

Asthma is thought to involve the influx of eosinophils ("eosinophilia") into the tissues of the lung (Frigas, E. et al., JA
llergy Clin. Immunol. 77: 527-537 (1986),
Incorporated herein by reference).

For the immunological basis of asthma, bronchoalveolar lavage studies (Godard, P. et al., J. Allergy Clin. Immunol. 70:88 (198
2)) and a study of respiratory smooth muscle tissue with exposed epithelium (Fla
vahan, NA et al., J. Appl. Physiol. 58: 834 (1985); Barne.
s, PJ et al., Br. J. Pharmacol. 86: 685 (1985)). Although these studies have not renamed the mechanism in immunology of asthma, they have led to generally accepted assumptions about the immunological cause of the disease (Frig
as, E. et al., J. Allergy Clin. Immunol. 77: 527-537 (198
6)).

Prominent pathological features of asthma are extensive eosinophil infiltration of lung parenchyma and disruption of mucociliary capacity. According to the so-called "eosinophil hypothesis", eosinophils are attracted to the bronchi to neutralize harmful mediators released from mast cells in the lung. According to this assumption, eosinophils are attracted to the bronchi, where degranulation takes place, releasing cytotoxic molecules. Upon degranulation, eosinophils release enzymes, such as histamine, allyl sulfatase and phospholipase D, which enzymatically neutralize noxious mediators of mast cells. However, these molecules also promote the destruction of the mucociliary mechanism,
Thus, it inhibits clearing of bronchial secretions, causing lung damage characteristic of asthma.

Since asthma involves the migration of cells, the use of an antibody that inhibits this migration has been proposed to mitigate the effects of allergens in the subject (WO 90/10453).

SUMMARY OF THE INVENTION The present invention is based on the discovery of a novel cell adhesion molecule called intercellular adhesion molecule-3 (ICAM-3). The present invention further provides functional derivatives of ICAM-3, anti-ICAM-3 antibodies, humanized anti-ICAM-3 antibody fragments of the antibodies, and
It also relates to other molecules that can bind to ICAM-3 and inhibit the biological function of ICAM-3.

The invention further includes the use of all of the above molecules in therapy and prophylaxis.

In particular, the present invention is substantially free of natural contaminants,
Includes ICAM-3 or a functional derivative thereof.

The present invention relates to a recombinant or synthetic DNA encoding or expressing ICAM-3 or a functional derivative thereof.
A method for obtaining a molecule is provided.

The present invention further provides antibodies, and particularly monoclonal antibodies, capable of binding to a molecule selected from the group consisting of ICAM-3 and a functional derivative of ICAM-3.

The present invention further provides a hybridoma cell capable of producing the above-mentioned monoclonal antibody.

The present invention includes a method for preparing a desired hybridoma cell producing an antibody capable of binding to ICAM-3 or a functional derivative of ICAM-3. The method includes the following steps.

(A) substantially pure ICAM-3, a cell expressing ICAM-3, a membrane expressing ICAM-3, ICAM-3 bound to a carrier,
Immunizing an animal with an immunogen selected from the group consisting of a peptide fragment of ICAM-3 or a peptide fragment of ICAM-3 bound to a carrier; (b) spleen cells isolated from the immunized animal; (C) transforming the fused spleen and myeloma cells into antibody-secreting hybridoma cells; (d) screening the hybridoma cells for anti-IC
A step of finding a hybridoma cell capable of producing an AM-3 antibody.

The invention also provides a method of modulating ICAM-3 mediated biological function of a cell. The method includes providing an effective amount of an ICAM-3 modulator to a subject in need of such treatment. Here, the ICAM-3 modulator is an antibody capable of binding to ICAM-3; a fragment of the antibody, which is a fragment capable of binding to ICAM-3;
3; a functional derivative of ICAM-3; and ICAM-1, ICAM-
2, or ICAM-3 other than molecules of the CD-18 family
Are selected from the group consisting of:

The invention further provides a method of treating specific inflammation in humans and other mammals. The method includes providing a subject in need of such treatment with an anti-inflammatory agent in an amount sufficient to suppress inflammation. Here, the anti-inflammatory agent is an antibody capable of binding to ICAM-3; a fragment of the antibody, which is capable of binding to ICAM-3; substantially pure ICAM-3; Derivative; or selected from the group consisting of non-immunoglobulin antagonists of ICAM-3. Here, the inflammation is the result of a solid organ transplant (eg, a kidney), a non-solid organ transplant (eg, a bone marrow), or a tissue transplant.

The invention also provides a method for treating non-specific inflammation in humans and other mammals. The method includes providing a subject in need of such treatment with an anti-inflammatory agent in an amount sufficient to suppress inflammation. Here, the anti-inflammatory agent is an antibody capable of binding to ICAM-3; a fragment of the antibody, which is capable of binding to ICAM-3; substantially pure ICAM-3; Derivative; or selected from the group consisting of non-immunoglobulin antagonists of ICAM-3.

The invention further includes a method of treating inflammation as described above, where the inflammation is associated with the following conditions: That is, symptoms include adult respiratory distress syndrome; multiple organ dysfunction syndrome resulting from sepsis, trauma, or bleeding; reperfusion injury of myocardium or other tissues; acute glomerulonephritis; reactive arthritis; skin with acute inflammatory components Disease; other central nervous system inflammatory diseases such as acute purulent meningitis or stroke; heat injury; hemodialysis;
Selected from the group consisting of leukocyte-depleted blood transfusion; ulcerative colitis; focal ileitis; necrotizing colitis; granular transfusion-related syndrome; and cytokine-induced toxicity.

The present invention also includes a method for inhibiting metastasis of hematopoietic tumor cells. The cells utilize members of CD-18 (especially LFA-1) for migration. The method includes providing a patient in need of such treatment with an amount of the agent sufficient to inhibit metastasis. Here, the drug is an antibody capable of binding to ICAM-3; a toxin derivative of the antibody (deriva
tization); a fragment of the antibody, ICAM-3
A toxin derivative of the fragment; a substantially pure ICAM-3; a toxin-derivatized ICAM-3; a functional derivative of ICAM-3; a toxin derivatization of a functional derivative of ICAM-3; Alternatively, it is selected from the group consisting of non-immunoglobulin antagonists other than members of the CD-18 family of molecules.

The present invention also includes a method for inhibiting the growth of a tumor cell expressing ICAM-3. The method includes providing a patient in need of such treatment with an amount of the agent sufficient to inhibit proliferation. Here, the agent is an antibody capable of binding to ICAM-3; a toxin derivative of the antibody; a fragment of the antibody, which can bind to ICAM-3; a toxin derivative of the fragment; Selected from the group consisting of a functionalized derivative of a member of the CD-18 family molecule, or a toxin-derivatized member of the CD-18 family molecule.

The present invention also provides a method for detecting the presence of a cell expressing ICAM-3. The method includes the following steps.

(A) incubating a cell or cell extract in the presence of a nucleic acid molecule, wherein the nucleic acid molecule is a molecule capable of hybridizing to ICAM-3MRNA; Determining whether it has hybridized to a complementary nucleic acid molecule present in the cell extract.

The present invention also relates to ICAM-3 in biological fluid samples.
Provide a method for detecting the presence of The method includes the following steps.

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

The present invention also provides a pharmaceutical composition comprising:
(A) an antibody capable of binding to ICAM-3; a fragment of the antibody, which is capable of binding to ICAM-3; substantially pure ICAM-3; a functional derivative of ICAM-3; ICAM-, other than members of the 18 family of molecules
An agent and an immunosuppressant selected from the group consisting of 3 non-immunoglobulin antagonists.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Adhesion of Cell Lines to Purified LFA-1 Adhesion of cell lines (A) and lymphocytes (B) to purified LFA-1 was determined by ICAM-1, ICAM-2, and ICAM. -3
caused by. LFA-1, ICAM-1, ICAM-2, and ICA
LFA- in the presence of M-3 specific blocking MAb
1 Binding of BCECF labeled cells on coated microtiter wells. Control wells are coated without LFA-1.

Figure 2. Flow cytometric analysis of ICAM-1, ICAM-2, and ICAM-3 expression in cells. (A) Lymphoblastoid cell lines were transformed with saturating amounts of MAb RR1 / 1.
(Anti-ICAM-1, MAb CB-IC2 / 1 (anti-ICAM-2), MAb C
Labeled with either BR-IC3 / 1 (anti-ICAM-3) or unbound control MAb X63 (thin line), followed by FITC
-Labeled with anti-mouse immunoglobulin. (B) Resting human leukocytes and lymphocytes activated with 3-day PHA were analyzed for ICAM expression.

Figure 3. Immunoprecipitation of ICAM-3. (A) ¹²⁵ I-labeled cell lysates were immunoprecipitated with either unbound control X63 or MAb CB-IC3 / 1 (anti-ICAM-3). (B) ²⁵ I-labeled SKW3 cell lysate was treated with (+) or without (-) N-glycanase and unbound control MAb X63, MAb W6 / 32
(Anti-HLA-A, B, C), MAb RR1 / 1 (anti-ICAM-1), M
Ab CBR-IC2 / 1 (anti-ICAM-2) or MAb CB-IC3
/ 1 (anti-ICAM-3).

Figure 4. Immunoprecipitation of different ICAM-3s from ICAMs from lysates of ¹²⁵ I-labeled lymphocytes and neutrophils
-3 was immunoprecipitated using mAb-conjugated Sepharose.
The immunoprecipitated ICAM-3 was separated by polyacrylamide gel electrophoresis. Other mAbs were used to immunoprecipitate other cell surface molecules. Human Ig Sepharose (control lane), CBR-IC2 / 1 (ICAM-2), CBR-IC3
/ 1 (ICAM-3), TS2 / 4 (LFA-1), LM2 / 1 (MAC-
1).

Figure 5. PM for SKW3 binding to ICAM-1 and ICAM-3
Effect of A 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 in the literature (Dustin et al., Nature 341: 6).
19 (1989); Marlin et al., Cell 51: 813-819 (1987)).
One representative result is shown. The error line indicates the standard deviation (SD).

Figure 6. Blocking of PMA-stimulated SKW3 cell adhesion by antibodies PMA-stimulated SKW3 cells purified from ICAM-1 or ICA
Various antibodies were tested for their ability to block binding to M-3 as described in the literature (Dustin
Et al., Nature 341: 619 (1989); Marlin et al., Cell 51: 813-
819 (1987)). One representative result is shown. The error line indicates the standard deviation (SD).

Figure 7. Binding of COS transductants to purified ICAM's. COS cells were transfected with the LFA-1 expression vector as described in the literature (deFougerolles et al., J. Exp. M.
ed. 175: 185-190 (1992)). Anti-LFA-1 antibody (TS1 / 22)
The transfected cells were tested for their ability to bind to immobilized ICAM-1 and ICAM-3 in the presence or absence of. One representative result is shown. The line indicating the error indicates SD.

Figure 8. Temperature dependence of PMA-stimulated SKW3 binding to ICAM PMA-stimulated SKW at 4 ° C, 16 ° C, 22 ° C, and 37 ° C
Three cells were tested for their ability to bind to purified ICAM-1 and ICAM-3 as described in the literature (Marlin
Cell 51: 813-819 (1987)). The assay was performed with LFA-1
The test was performed in the presence or absence of the mAb (TS1 / 22).
One representative result is shown. The error line is SD
Is shown.

Figure 9. Antibody blocking of PHA-stimulated T cell division by antibodies. Various antibodies were tested for their ability to block PHA-stimulated T cell division as described in the literature (Krensky et al., J. Immunol. 131: 611-616 (1983)).

Figure 10. Effect of anti-ICAM antibodies on mixed lymphocyte responses. Various antibodies were tested for their ability to block mixed lymphocyte responses as described in the literature (Krensk
y et al., J. Immunol. 131: 611-616 (1983)).

Figure 11. Gas Chromatography of a Peptide Fragment of ICAM-3 ICAM-3 was purified and digested with Lys-C. Peptide fragments were separated by high performance liquid chromatography.

Figure 12. Schematic of clone 11.2 Schematic of clone 11.2. The positions of the NK-10 and NK-17 peptides, the various DNA sequences obtained, and the transmembrane regions are indicated.

Figure 13. Comparison of the NK-10 primary sequence with human ICAM-1. To identify the homology between the NK-10 primary sequence and the human ICAM-1 protein, the GAP program was used.

Figure 14. Sequence comparison between RM13 primary sequence and human ICAM-1. To identify sequence homology between RM13 primary sequence and human ICAM-1, the GAP program was used.

Figure 15. Sequence comparison between RM13 primary sequence and human ICAM-2. To identify sequence homology between RM13 primary sequence and human ICAM-2, the GAP program was used.

Figure 16. Sequence comparison between T7 primary sequence and human ICAM-1. To identify sequence homology between T7 primary sequence and human ICAM-1, the GAP program was used.

Figure 17. Sequence comparison between T7 primary sequence and human ICAM-2. To identify sequence homology between T7 primary sequence and human ICAM-2, the GAP program was used.

Fig. 18. Partial sequence of ICAM-3 The DNA sequence of clone 11.2 was determined by a conventional method.

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

B. Sequence obtained from the ICAM-3 gene. These sequences were obtained by priming clone 11.2 with the NK-10 probe.

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

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is based on the discovery of a previously unidentified binding ligand for LFA-1. Molecules such as the CD-18 family that are involved in the process of cell adhesion are termed "adhesion molecules".

I. LFA-1 The leukocyte adhesion molecule LFA-1 is used in a wide range of lymphocytes, monocytes, natural killer cells,
And mediates the interaction of granulocytes with other cells (Spri
nger, TA et al., Ann. Rev. Immunol. 5: 223-252 (198
7)).

LFA-1 is a receptor for ICAM-1, ICAM-2, and newly identified ICAM-3, which is disclosed herein. These surface molecules are constitutively expressed in some tissues and are induced by inflammation in other tissues (Marlin, SD et al., Cell 51: 813-819 (1987); Du
stin, ML et al., J. Immunol. 137: 245-254 (1986); Dusti.
n, ML et al., Immunol. Today 9: 213-215 (1988); 1987.
U.S. Patent Application No. 07 / 019,440, filed February 26, 2016
No., and U.S. patent application Ser.
07 / 250,446, both applications are incorporated herein by reference).

LFA-1 functions in the interaction of antigen-specific and antigen-independent T cytotoxicity, T-perper, natural killers, granulocytes, and monocytes on other cell types (S
Pringer, TA et al., Ann. Rev. Immunol. 5: 223-252 (198
7); Kishimoto, TK et al., Adv. Immunol. (1988, printing)).

II. ICAM-1 ICAM-1 has a size of 76-114k on different cells.
D is a single-chain glycoprotein that is a member of the Ig superfamily with five C-like domains (Dustin, ML et al., Immunol. Today 9: 213-215 (1
988): Staunton, DE et al., Cell 52: 925-933 (1988): Si.
mmons, D. et al., Nature 331: 624-627 (1988)). ICAM−
1 can be highly induced on a wide range of cell types by cytokines including IFN-g, TNF, and IL-1 (Dust
in, ML 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, ML et al., J. Immunol. 137: 245-254).
(1986); Dustin, ML et al., J. Cell. Biol. 107: 321-331.
(1988); Dustin, ML et al., J. Exp. Med. 167: 1323-1340.
(1988)). Adhesion is blocked by pretreating lymphocytes with LFA-1 MAb or by pretreating other cells with ICAM-1 MAb (Dustin, ML et al., JI.
mmunol. 137: 245-254 (1986); Dustin, ML et al., J. Cell.
Biol. 107: 321-331 (1988); Dustin, ML et al., J. Exp. Me.
d.167: 1323-1340 (1988)). Similar results were obtained using purified ICAM-1 in artificial membranes or on petri dishes, indicating that LFA-1 and ICAM-1 are receptors for each other (Marlin, SD et al., Cell 51: 8
13-819 (1987); Makgoba, MW et al., Nature 331: 86-88.
(1988)). For distinction, they are called "receptor" and "ligand", respectively. For ICAM-1, US patent application Ser. No. 07 / 045,963; 07/115, 79
No. 8; 07/155, 943; 07/189, 815 or 07 /
It is also described in No. 250, 446, these are for reference
All are incorporated herein by reference. III. ICAM-2 The existence of another LFA-1 ligand from ICAM-1 was expected (Rothlein, R. et al., J. Immunol. 137: 1270-1274 (1
986); Makgoba, MW et al., Eur. J. Immunol. 18: 637-640 (1
988); Dustin, ML et al., J. Cell. Biol. 107: 321-331 (198
8)). The second LFA-1 ligand identified was "ICAM-
2 ".

ICAM-2 differs from ICAM-1 in cell distribution and lack of cytokite induction. I c
AM-2 is an integral membrane protein with two Ig-like domains, while ICAM-1 has five Ig-like domains (Staunton, DE et al., Cell 52: 925-933 (198
8): Simmons, D. et al., Nature 331: 624-627 (1988)).
In particular, ICAM-2 is highly related to the two most N-terminal domains of ICAM-1 (34% identical), indicating that this proportion indicates that ICAM-1 or ICAM-2 is a member of another Ig superfamily. Greater for members. From this,
It turns out to be a subfamily of Ig-like ligands that bind to the same integrin family receptor. ICAM−
No. 2 is also described in US patent application Ser. No. 07 / 454,295, which is incorporated herein by reference.

IV. ICAM-3 The present invention relates to a third LFA-1 ligand, "ICAM-
3). The development of MAbs against ICAM-2 analyzed the previous LFA-1-dependent, ICAM-1-independent phenomena, and suggested that a third ligand for LFA-1 was present. Binding of several cell types, such as epithelial and endothelial cells, to purified LFA-I
-1 and ICAM-2 MAbs can be completely blocked, while ICAM-1, ICAM-2 independent LFA-
The pathway for 1 also exists in many lymphoid cell lines, including the T cell lymphoma cell line SKW3 (FIG. 1).

To investigate the characteristics of this adhesion pathway, MA for SKW3
b was screened for the ability to inhibit this cell line from binding to LFA-1 with anti-ICAM-1 and anti-ICAM-2 MAbs. One MAb, CBR-IC3 / 1, was selected that completely inhibited this novel adhesion pathway (FIG. 1). The adhesion of SKW3 to purified LFA-1 was only slightly inhibited by the combination of blocking anti-ICAM-1 and anti-ICAM-2 MAb. Anti-ICAM-3MAb CB-IC3 /
Addition of 1 alone inhibited adhesion well and was completely inhibited by the combination with blocking anti-ICAM-2 MAb.
Thus, adhesion of SKW3 to purified LFA-1 was mostly mediated by ICAM-3 and in part by ICAM-2. Because of the different pattern of MAb inhibition, LFA
In adhesion to -1, each of the four cell lines
Different proportions of these three ICAMs were used. Adhesion of JY in the B lymphoblastoid cell line occurs primarily by the ICAM-1 pathway and only slightly by the ICAM-2 and ICAM-3 pathways. The SLA of other B lymphoblastoid cell lines used both ICAM-1 and ICAM-3. This is because the adhesion is ICAM-
It was shown that either 1 MAb or ICAM-3 MAb was not inhibited alone, but was almost completely inhibited when both MAbs were used together. The thymoma cell line, Jurkat,
Both ICAM-2 and ICAM-3 adhesion pathways were used and were slightly dependent on ICAM-1. The adhesion of resting T lymphocytes and phytohemagglutinin (PHA) activated T lymphocytes was also examined (FIG. 1B). Resting lymphocytes have been described in the literature to bind strongly to purified LFA-1 (Dustin et al., Nature 341: 619-624 (1989)) and this binding is highly dependent on ICAM-3. I understand. Activation by PHA induces ICAM-1 expression and adhesion to LFA-1 is mainly due to ICAM-1
And partly due to ICAM-3. The ICAM-2 component of adhesion in both resting and activated T cells was detectable but masked by the presence of ICAM-1 and ICAM-3. For each cell, to achieve substantial inhibition, MA against at least two ICAMs
Since b was needed, using ICAM requires a lot of effort. The notable exception to this is the resting lymphocyte LF
A-1 is an adhesion to A-1.
-3 was mediated. In all cases, all three combinations of anti-ICAM-MAbs eliminate binding to LFA-1 to levels comparable to those seen in the presence of anti-LFA-1 MAb.

The relative affinities of the three ICAMs for LFA-1 are compared to the contribution of ICAMs to LFA-1 examined above and the cell surface expression of ICAMs measured by immunofluorescence flow cytometry. (FIG. 2). Comparison of the surface expression of ICAM with its contribution to LFA-1 adhesion indicates that ICAM-1 has a very high affinity for LFA-1 and that ICAM-2 and ICAM-
3 was found to have similar and low affinity.
For example, Jurkat cells expressed ICAM-2 and ICAM-3 at the same level and each contributed to binding to LFA-1. In cases such as JY cells where ICAM-2 expression is much greater than ICAM-3, the ICAM-2 pathway of LFA-1 adhesion is
Was better. In contrast, SLA and SKW3 are
It expressed 3 to 5 times as much ICAM-3 as M-2, and, of course, the ICAM-3 pathway of adhesion was superior to the ICAM-2 pathway. ICAM-3 was well expressed on resting T lymphocytes, without ICAM-1, and adhesion to LFA-1 was mainly mediated by ICAM-3. In cases such as SLA, JY, and PHA-activated T cells where ICAM-1 is well expressed, ICAM-1
-1 is the main route of adhesion.

The pattern of distribution of ICAM-3 was ICAM-1 and ICAM-
It was somewhat different from the two. ICAM-1 and ICAM-2
In contrast, ICAM-3 was not expressed on either resting or stimulated endothelial cells (data not shown). This is consistent with the finding that LFA-1-dependent binding of cells to both resting or stimulated endothelial cells is an ICAM-1- and ICAM-2-dependent phenomenon. ICAM-3, with some exceptions, is restricted to the hematopoietic system and was highly expressed in lymphoid and monocyte-like cell lines. However, in all cases, the expression of ICAM-3 on the cell line was
-1 dependent and cooperated with ICAM-1 and ICAM-2 independent adhesion pathways. LF exclusively by ICAM-1 and ICAM-2
Cell lines that bind to A-1 (HUVEC, Raji) show no expression of ICAM-3, whereas cell lines that show weak binding by this third pathway of adhesion (JY, U937, SupT) The surface expression of ICAM-3 was low (data not shown).

ICAM-3 is characterized by its expression on leukocytes, ICAM-1 and ICAM-1.
It was significantly different from ICAM-2 (FIG. 2B). ICAM-3
High levels were expressed on resting lymphocytes, monocytes and neutrophils, while ICAM-1 and ICAM-2 were very low or absent. By comparison, ICAM-1
And ICAM-2 was slightly expressed on monocytes, and only ICAM-2 was present on resting lymphocytes. Neither ICAM-1 nor ICAM-2 was expressed on neutrophils. Activation of lymphocytes by PHA increased ICAM-3 expression by a factor of 2-3 and also induced ICAM-1 expression significantly (Dustin et al., J. Immunol. 137: 245-254).
(1986)) (Figure 2B).

Immunoprecipitates of ICAM-3 from various ¹²⁵ I-labeled cell lines revealed a sharp band of 124,000 Mr under reducing conditions and a slight increase in mobility under non-reducing conditions (FIG. 3A). Upon treatment with N-glycanase, the ICAM-3 band was reduced to Mr 87,000 and ICAM-
3 was a highly glycosylated protein like ICAM-1 and ICAM-2 (FIG. 3).
B). The biological characteristics, pattern of expression, and functional properties of ICAM-3 distinguish it from previous adhesion molecules. Previous adhesion molecules include the human homing receptor LAM-1 (Tedder et al., J. Immunol. 144: 532-54).
0 (1990)), the inducible endothelial adhesion molecule VACAM-1 (Rice
J. Exp. Med. 171: 1367-1374 (1990); Carlos et al., Blo.
od printing (1990); (Osborn et al., Cell 59: 1203-1211.
(1989)), and the VLA family of matrix receptors (Hemler, ME, Ann. Rev. Immunol. 8: 365-400 (19
90)). No MAb with a similar cell distribution was found in the fourth leukocyte workship database (Gilks, WR et al., Leukocyte Typing Data Ba
se IV, Oxford University Press, Oxford, England (199
0)).

The presence of the three LFA-1 ligands suggests different aspects of LFA-1-dependent leukocyte interactions.
ICAM-1 is basically expressed on endothelial cells, and on many types of epithelial cells, and is strongly induced in inflammation and immunity, regulates cell positioning, and has specific antigen recognition (Wawryk et al., Immuno
l. Rev. 108: 135-161 (1989)). Because ICAM-2 is the predominant LFA-1 ligand on resting endothelial cells, this pathway of adhesion depends on normal LF by tissue endothelial cells.
It can have a significant effect on the recirculation of lymphocytes bearing A-1 (Hamann et al., J. Immunol 140: 693-699 (1988); Mac)
Kay et al., J. Exp. Med. 171: 810-817 (1990); Pals et al., J. Im.
munol 140: 1851-1853 (1988); and (Nunoi et al., Hu
m.Path. 19: 753-759 (1988)). Currently, the function of ICAM-3 on neutrophils remains unknown. because,
Despite the presence of LFA-1, homotypic aggregation of neutrophils appears to be primarily Mac-1 dependent (And
erson et al., J. Immunol. 137: 15-27 (1986); Patarroyo
Et al., Scand. J. Immunol 22: 619-631 (1985)). The discovery that resting T lymphocyte adhesion to LFA-1 is primarily caused by ICAM-3 and that ICAM-3 is more well expressed on monocytes and resting lymphocytes compared to other LFA-1 ligands. This fact, combined with the facts, suggests an important role in the initiation of the immune response. Indeed, adhesion of T cells to antigen presenting cells requires an LFA-1: ICAM interaction (Dra
nsfield et al., Imm. Rev. 114: 29-44 (1990);
mmunol. Today 10: 417-422 (1989)). And especially IC
On resting T lymphocytes that express neither AM-1 nor ICAM-2, ICAM-3 itself may play a role. In fact,
A role for LFA-1 ligands other than ICAM-1 in allogeneic and autologous mixed lymphocyte reactions has been proposed (Bagnasc
o et al., Cell Immunol. 128: 362-369 (1990)). further,
ICAM-3 is expected to be important in antigen-specific interactions between T and B lymphocytes. here,
One of these cells is in an unactivated state. ICAM-3 also depends on LFA-1 but has
May be important for the lysis of specific targets by T cells independent of CAM-1 (Makgoba et al., Eur. J. Immunol 18: 637).
-640 (1988)).

The presence of multiple ICAMs has important implications for clinical treatment of ICAMs or ICAM analogs with MAbs. ICAM-1 MAb is expressed in vivo in the kidney (Cosimi
J. Immunol. 144: 4604-4612 (1990)) and cardiac (Flavin et al., Transplant. Proc. 23: 533-534 (1991)).
It is effective in prolonging allogeneic transplantation. ICAM-3MAb
Inhibits a different and overlapping number of immune responses in vivo because it inhibits LFA-1-dependent adhesion interactions of a subset of different cell types.

V. cDNA Cloning of ICAM-3 Any of a variety of methods can be used to clone the ICAM-3 gene. In one such method,
Shuttle vector library for cDNA inserts (ICAM
-3 expressing cells) from the presence of an insert containing the ICAM-3 gene. Such an analysis can be performed by transfecting cells with the vector and assaying for ICAM-3 expression.

The ICAM-3 cDNA is preferably prepared by the method of Aruffo and Seed for identifying ligands for adhesion molecules (Seed, B. et al., Proc.
c. Natl. Acad. Sci. USA 84: 3365-3369 (1987)). In this method, a cDNA library is prepared from cells expressing ICAM-3 (such as SLA, Jurkat, or SKW3 lymphoblastoid cell line). Preferably, a cDNA library is prepared from T cells. This library is typically used to transfect cells that do not express ICAM-3 (eg, Cos cells or HeLa cells). The transfected cells are placed in a Petri dish previously coated with LFA-1 or anti-ICAM-3 antibody. Transfected cells containing the sequence encoding ICAM-3 and expressing the ligand on the cell surface were LFA-1 or anti-ICAM on the Petri dish surface.
Adhere to -3 antibody. The non-adherent cells are washed away, and then the adherent cells are removed from the Petri dish and cultured. Recombinant ICAM
Determine the sequence of the-3 expressing cells.

When LFA-1 was used for coating petri plates, I
Anti-ICAM-1 and anti-ICAM-2 specific antibodies are added to Petri dishes to prevent adhesion of CAM-1 or ICAM-2 expressing cells. LF of ICAM-1 ⁺ or ICAM-2 ⁺ transductants
A-1 Adhesion to coated plastic is thus RR1 /
1, can be inhibited by antibodies such as anti-ICAM-1 MAb and CBR-IC2 / 2, anti-ICAM-2 MAb. Binding of cells transfected with ICAM-3 to LFA-1 coated Petri plates is inhibited by EDTA and anti-LFA-1 MAb, but not by anti-I
It is not inhibited by CAM-1 or anti-ICAM-2 MAb. Therefore, cells expressing ICAM-1 or ICAM-2 cannot adhere to the Petri dish and are washed away with almost no other adherent cells. As a result, ICAM-3
Are expressed are enriched.

Other methods for obtaining the gene sequence encoding ICAM-3 are well known in the art, and those skilled in the art routinely use these methods to obtain the desired gene.

One such method for obtaining a gene sequence encoding ICAM-3 is to use an oligonucleotide probe to screen a cDNA library or a chromosomal DNA library.
In this method, the ICAM-3 protein is preferably purified using immunopurification methods well known in the art, and the terminal amino acid sequence is determined by methods known in the art. Alternatively, ICAM-3 is replaced with trypsin or Ly
The peptide is purified by digestion with an enzyme such as sC, and the amino acid sequence of one of the internal fragments is determined.

Once the partial amino acid sequence has been determined, oligonucleotide probes are created based on the preferred codons for the organism, but degenerate probes are created based on all possible codon combinations, or preferred codons are known. Using a combination of ICAM-1 or ICAM-2 DNA sequence homology and codon degeneracy,
Create a probe. This probe is used to screen a chromosome library or a cDNA library, and a sequence that hybridizes to the probe is found.

By a technique as described above or similar thereto, the gene for human aldehyde dehydrogenase (Hsu, LC et al.,
Proc. Natl. Acad. Sci. USA 82: 3771-3775 (1985)), the gene for fibronectin (Suzuki, S. et al., Eur. Mol. Biol.
Organ. J. 4: 2519-2524 (1985)), human estrogen receptor gene (Walter, P. et al., Proc. Natl. Acad. Sci. US
A 82: 7889-7983 (1985)), and a tissue-type plasminogen activator (Pennica, D et al., Nature 301: 214).
−221 (1983)) was successfully cloned.

In yet another ICAM-3 gene cloning method, an expression library is prepared by cloning chromosomal DNA or, more preferably, cDNA from ICAM-3 expressing cells. Next, this library is
-3 Antibody is screened for a member capable of expressing a protein that binds thereto.

The cloned ICAM-3 gene obtained by any of the above methods is operably linked to an expression vector and introduced into a bacterium or eukaryotic cell,
ICAM-3 protein can be produced. Techniques for such manipulations are disclosed in Maniatis, T. et al., Supra, and are well known in the art.

In the method using PCR for cloning the ICAM-3 gene, ICAM-3 is used for ICAM-1 and / or ICAM-1.
It is assumed that there is homology with -2. ICAM-1
Amplification of cDNA or mRNA by polymerase chain reaction is performed using a degenerate oligonucleotide based on a sequence conserved between ICAM-2 and ICAM-2. This cDNA or mRNA is derived from cells such as SKW3 or tonsil, which are known to express ICAM-3 protein (deFougerolles et al., J. Exp. Med., 175: 18).
5-190 (1992)). The clone was sequenced and ICAM
Different from ICAM-1 and ICAM-2 are used to obtain full-length cDNA clones.

In all of the above methods, the authenticity of the clone was confirmed by expressing the full-length clone, for example, in COS cells and examining its reactivity with ICAM-3 mAb.

VI. Agents of the Invention: ICAM-3 and functional derivatives thereof,
Agonists and Antagonists The present invention further provides ICAM-3, "functional derivatives" thereof, "agonists" and "antagonists".
With the goal.

A. Functional Derivatives of ICAM-3 A “functional derivative” of ICAM-3 is defined as a biological activity (either functional or structural) that is substantially similar to the biological activity of ICAM-3. Compounds. The term "functional derivative" includes "fragments,""variants," and "chemical derivatives" of the original ICAM-3 molecule.

A “fragment” of ICAM-3 refers to any subset of the polypeptides of this molecule. ICAMs that have ICAM-3 activity and are soluble (ie, do not bind to membranes)
The -3 fragment is particularly preferred. Soluble fragments are preferably made by deleting regions spanning the membrane of the original molecule, or by substituting or deleting hydrophilic amino acid residues instead of hydrophobic amino acid residues. Identification of such residues is
It is well known in the art.

Fragments containing some of the complete Ig-like domains such as domain 1 (D1), D1 and D2, D1-3, D1-4, and D1-5 are preferred.

A "variant" of ICAM-3 refers to a molecule having a structure and function substantially similar to either the complete molecule or a fragment thereof.

A molecule is “substantially similar” to another molecule,
Either both molecules have substantially similar structures or both molecules have similar biological activities. Thus, if two molecules have similar activities, they are considered variants as referred to herein. This is also the case when one molecule has a structure that cannot be found in the other molecule, or when the amino acid residue sequences are not identical.

In the present invention, a molecule is a "chemical derivative" of another molecule if it contains additional chemical components not normally found in the native molecule. Such components may improve the solubility, absorption, biological half-life, etc. of the molecule. These components may alternatively reduce the toxicity of the molecule or eliminate or attenuate any undesirable side effects of the molecule. Components that can mediate such effects include Remington's Pharma
ceutical Sciences (1980).

"Toxin-derivatized" molecules constitute a special class of "chemical derivatives".

A “toxin derivatized” molecule is a molecule (such as an ICAM-3 or anti-ICAM-3 antibody) covalently linked to a toxin component. Methods for attaching such components to molecules are well known in the art.

The binding of such a molecule to the cell causes the toxin component to be delivered in close proximity to the cell, thereby promoting cell killing. All suitable toxin components can be used. However, for example, ricin toxin, cholera toxin, diphtheria toxin, radioisotopotoxin, or membrane channel forming toxin are preferably used.

Functional derivatives of ICAM-3 of up to about 100 residues can be conveniently prepared by in vitro synthesis. If desired, such fragments can be modified using methods known in the art. The modification is performed by reacting a target amino acid residue of the purified or crude protein with an organic inducer capable of reacting with a specific side chain or terminal residue. The resulting covalent derivative can be used to identify residues important for biological activity.

Many methods can be used to make and isolate fragments of ICAM-3. ICAM-3 can be cross-linked to a water-insoluble support matrix using conjugation with a bifunctional agent. Alternatively, reactive water-insoluble matrices such as hydrocarbons activated with cyanogen bromide, and U.S. Patent Nos. 3,969,287; 3,691,016;
No. 4,195,128; No. 4247,642; No. 4,229,537; and
The reactive substrate described in 4,330,440 is used for protein immobilization.

A functional derivative of ICAM-3 having an altered amino acid sequence can also be prepared by mutating the DNA encoding ICAM-3. Such functional derivatives include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence of ICAM-3. As long as the final construct has the desired activity, any combination of deletions, insertions, and substitutions
Can be used to make the final construct. Obviously, mutations in the DNA encoding the functional induction should not be placed in sequences outside of the reading frame and, preferably, create complementary regions that make up the secondary structure of the mRNA. Not created (EP Patent Application Publication No. 75,4
No. 44).

At the genetic level, functional derivatives are obtained by site-directed mutagenesis of the DNA encoding ICAM-3, and thereby the production of DNA encoding the functional derivative, and subsequent expression of the DNA in recombinant cell culture. Can be prepared. The site for introducing a mutation in the amino acid sequence is determined in advance, but the mutation itself need not be determined in advance. For example, random mutagenesis, such as linker scanning mutagenesis, can be performed at the target codon or target region to properly make mutations at defined locations, producing many derivatives. The derivative is then expressed and the appropriate combination of desired activities is screened. Techniques for creating mutations at predetermined sites in a known DNA sequence are well known, for example, site-directed mutagenesis.

The preparation of the functional derivative of ICAM-3 in the present specification is preferably carried out by site-directed mutagenesis of ICAM-3 or the DNA encoding the functional derivative of ICAM-3 prepared above. The technique of such site-directed mutagenesis,
Well known in the art, Maniatis, T. et al; Molecula
r Cloning, a Laboratory Manual, Coldspring Harbor, NY
(1982), the disclosure of which is incorporated herein by reference. Site-directed mutagenesis involves the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutation,
A functional derivative of AM-3 can be made.

Amino acid deletions, insertions, or substitutions can be made using site-directed mutagenesis. Amino acid sequence deletions are generally from about 1 to 30 residues, more preferably 1 to 10 residues, typically
Each is adjacent. The most preferred defect is ICAM-3
In order to make a soluble form of ICAM−
The soluble form of 3 is most preferably made by deleting either the membrane-widening region of ICAM-3 or the hydrophobic region in ICAM-3.

Amino acid insertions include inserting one or more amino acid residues in the ICAM-3 coding sequence and fusing terminally with a polypeptide of virtually unlimited length. Insertion into the sequence (eg, complete ICAM-3
Insertion into the molecular sequence) may generally range from about 1 to 10 residues, more preferably 1 to 5 residues. Examples of terminal insertions include fusion of one sequence, heterologous or homologous to the host cell, with the N-terminus of the molecule to facilitate secretion of the derivative from the recombinant host.

A third group of functional derivatives comprises one of the ICAM-3 molecules.
The above amino acid residues have been removed, and a different residue has been inserted in its place. Such substitutions are preferably made according to the following table if it is desired to fine-tune the properties of the ICAM-3 molecule.

Table 1 Original residue substitution sequence Ala gly; ser Ara lys Asn gln; his Asp glu Cys ser Gln asn Glu asp Gly ala; pro His asn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Met leu; tyr; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu Substantial changes in functional or immunological properties are shown in Table 1.
By selecting substitutions that are less conservative than those described in. That is, (a) the backbone structure of the polypeptide in the region of substitution, eg, sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the conservation of the side chain moiety. This is done by selecting the influencing residues. Generally, less conservative substitutions include (a)
Replacing, deleting or inserting glycine and / or proline with another amino acid; (b) replacing a hydrophilic residue with a hydrophobic residue; (c) replacing a cysteine residue with any other residue Or place another residue in place of the cysteine residue; (d) replace the residue with an electropositive side chain with a residue with an electronegative charge or replace the electropositive side chain Placing a residue with an electronegative charge in place of the residue having
Or (e) replacing a residue having a large side chain with a residue having no such side chain, or replacing a residue having a large side chain with a residue having no such side chain. Put, say the thing.

Most preferred are substitutions that affect the solubility of ICAM-3. This is done by substituting hydrophilic amino acid residues for hydrophobic amino acids.

Mutants designed to increase the affinity of ICAM-3 can be made by introducing amino acids present at positions similar to ICAM-1 or ICAM-2. Similarly, IC
Mutant ICAM-3 molecules lacking N-linked CHO at positions similar to AM-1 or ICAM-2 can be prepared.

It is difficult to accurately predict how a particular substitution, deletion, or insertion affects the biological activity of ICAM-3 before actually doing it. However, it will be apparent to one skilled in the art that such effects can be evaluated in routine screening assays. For example, derivatives are typically made by linker scanning site-directed mutagenesis of DNA encoding the native ICAM-3 molecule. This derivative is then expressed in a recombinant host and optionally purified from the cell culture, for example by immunoaffinity chromatography.

Next, the activity of the cell lysate or purified derivative is screened by a suitable screening assay to select those having desired properties. For example, the immunological properties of a functional derivative, such as affinity for a particular antibody, are measured by a competitive immunoassay. Changes in immunomodulatory activity are measured by a suitable assay. Redox or thermal stability, biological half-life, hydrophobicity,
Modification of the protein's properties, such as its susceptibility to proteolytic digestion or its tendency to aggregate with carriers or into multimers, is assayed by methods well known to those skilled in the art.

B. Agonists and Antagonists of ICAM-3 An “agonist” of ICAM-3 refers to a compound that enhances or increases the ability to exert any biological function of ICAM-3. Examples of such agonists are those that increase the ability of ICAM-3 to bind to cellular receptors.

An "antagonist" of ICAM-3 refers to a compound that reduces or prevents the ability of ICAM-3 to exert any biological function. Examples of such antagonists include ICAM-
1 and ICAM-2, functional derivatives of ICAM-1 and ICAM-2, anti-ICAM-3 antibodies, anti-LFA-1 antibodies and the like.

For cell agglutination assays, see U.S. patent application Ser.
Nos. 5,963; 07 / 115,798; 07 / 155,943; 07 / 189,815 or 07 / 250,446, all of which are incorporated herein by reference. Such an assay allows the measurement of LFA-1-dependent aggregation, thus
It can be used to identify agents that affect the extent of CAM-3 / LFA-1 aggregation. As such, this assay uses IC
It can be used to identify AM-3 agonists and antagonists. The antagonist is LFA-1 or I
It may act to impair the ability of CAM-3 to mediate aggregation.
In addition, non-immunoglobulin (ie, chemical) drugs
The assays described above can be used to determine whether an agonist or antagonist of ICAM-3 / LFA-1-mediated aggregation. Such agglutination assays typically involve PH
Performed using peripheral T cells stimulated with A. Therefore, binding of peripheral blood to LFA-1 is used to assay ICAM-3-mediated aggregation.

C. Anti-ICAM-3 Antibody A preferred immunoglobulin antagonist of the invention is
Disclosed herein, ICAM-3 such as CBR-IC3 / 1
Antibodies. Other suitable anti-ICAM-3 antibodies can be obtained in various ways.

Antibodies to ICAM-3 (or a functional derivative of ICAM-3) can be either polyclonal or monoclonal. In addition, the antibodies of the present application can be obtained by methods known in the art or by PCT Application Nos. PCT / US91 / 02942 and P.T.
It can be humanized by the method disclosed in CT / US91 / 02946.

An anti-ICAM-3 antibody can be prepared by introducing ICAM-3 or a peptide fragment thereof into an appropriate animal. The immunized animal will produce polyclonal antibodies against it. Using a peptide fragment of ICAM-3, a region-specific antibody can be obtained that reacts only with the epitope (s) contained in the peptide fragment used to immunize the animal.

Alternatively, anti-ICAM-3 antibodies can be made using ICAM-3, which is naturally expressed on the surface of lymphocytes. When such cells are introduced into an appropriate animal, such as by intraperitoneal injection,
Antibodies that can bind to ICAM-3 or CD-18 family molecules are produced. If desired, the serum of such animals can be removed and used as a source of polyclonal antibodies capable of binding to these molecules.

Alternatively, anti-ICAM-3 antibodies can be obtained from Selden, RF (EP-A-289,034) or Selden, RF et al., (Sc
ience 236: 714-718 (1987)). Specifically, cells of a suitable animal (eg, mouse) are
Transfect with a vector capable of expressing the complete ICAM-3 molecule, or a fragment thereof. ICAM-3 production in the transfected cells of the animal elicits an immune response in the animal, and the animal produces anti-ICAM-3
Antibodies are produced.

However, from animals immunized by any of the above methods,
Preferably, spleen cells are removed and monoclonal antibodies capable of binding to ICAM-3 are produced.

The hybridoma cells obtained as described above are screened by various methods to identify hybridoma cells secreting an antibody capable of binding to ICAM-3. In a preferred screening assay, such a molecule is ICAM-3
And the ability to inhibit aggregation of cells that do not express ICAM-1 and ICAM-2. Antibodies capable of inhibiting such aggregation are further screened for ICAM-
3 to inhibit such aggregation or to bind to members of the CD-18 family molecule to inhibit such aggregation. In such a screen, any method that can distinguish ICAM-3 from other CD-18 families can be used. Thus, for example, by immunoprecipitation and polyacrylamide gel electrophoresis of an antigen that can bind to the antibody,
Can be analyzed. Distinguishing these antibodies between antibodies that bind to molecules of the CD-18 family and antibodies that bind to ICAM-3 depends on the ability of the antibodies to bind to LFA-1 expressing cells but not to ICAM-3 expressing cells. It is possible by screening (or vice versa). The ability of an antibody to bind to LFA-1 expressing cells but not to ICAM-3 expressing cells can be detected by methods commonly used by those skilled in the art. Such methods include immunoassays (especially those using immunofluorescence), cell aggregation, filter binding studies, antibody precipitation, and the like.

In a preferred method, antibodies are selected that have the ability to bind to cells that express ICAM-3 and not to cells that do not express ICAM-3.

In addition to the above-mentioned ICAM-3 agonists and antagonists, in the present invention, other drugs that can be used for treatment of inflammation, HIV infection, asthma, etc. include anti-idiotype antibodies against anti-ICAM-3 antibodies, and receptor molecules Or fragments of such molecules that can bind to ICAM-3.

The anti-idiotypic antibodies of the invention can bind in (or exclude) competition with ICAM-3. Such antibodies, for example, elicit antibodies against anti-ICAM-3 antibodies,
It can then be obtained by screening for the ability of ICAM-3 to bind to one of the natural ligands.

Since molecules of the CD-18 family can bind to ICAM-3, such molecules (eg, heterodimers with both alpha and beta subunits,
Or molecules comprising only alpha or beta subunits, or molecules having fragments of both or one of the subunits).
It will compete with other cells for binding to ICAM-3 present on the cell.

Anti-aggregate antibodies of the invention can be identified and titered in a variety of ways. For example, the ability of the antibody to bind to ICAM-3 expressing cells (eg, T cells, etc.) and not bind to cells that do not express ICAM-3 can be measured. A suitable assay for cell aggregation is described in U.S. Patent Application No. 07 / 045,963.
No. 07 / 115,798; No. 07 / 115,943; No. 07 / 189,815
No. 07 / 250,446, all of which are incorporated herein by reference. Alternatively, the ability of the antibody to bind to ICAM-3 or a peptide fragment of ICAM-3 can be measured. As will be apparent to those skilled in the art, the above assays can be modified to provide a variety of potential screening assays,
Alternatively, they can be performed in a different order. These assays are based on antibodies that can bind to ICAM-3 and CD-
Antibodies that can bind to molecules of 18 family members can be identified and distinguished.

D. Preparation of Agents of the Invention Agents of the invention can be used to induce natural processes (eg, animals, plants, fungi, bacteria, etc.) to produce non-immunoglobulin antagonists of ICAM-3, or To produce a polyclonal antibody capable of binding to ICAM-3); synthetic (eg, ICAM-3).
-3, synthesizing a functional derivative of ICAM-3 or a protein antagonist of ICAM-3 (either immunoglobulin or non-immunoglobulin); hybridoma technology (for example, monoclonal antibodies capable of binding to ICAM-3; Or recombinant techniques (eg, using the agents of the invention in various hosts (ie, yeast, bacteria, fungi, cultured mammalian cells, etc.) using recombinant plasmids or viral vectors). Making the drug). The choice of which method to use depends on factors such as convenience, desired yield, and the like. However, it is not necessary to use only one of the above methods, processes, or techniques to make a particular anti-inflammatory agent, and the above methods, processes, or techniques may be combined in obtaining a particular drug. be able to.

VII. Use of ICAM-3 and Functional Derivatives, Agonists, and Antagonists The agents of the present invention can be used to modulate various biological activities mediated by ICAM-3.

A. Inhibition of Inflammation One of the present invention is derived from the ability of ICAM-3 and its functional derivatives to bind to molecules of the CD-18 family, especially LFA-1. ICAM-3 suppresses (ie, prevents or attenuates) inflammation because it can interact with members of the CD-18 family of glycoproteins.
Can be used to

As used herein, the term “inflammation” includes both specific and non-specific defense system responses.

As used herein, the term “specific defense system” refers to a component of the immune system that responds to the presence of a specific antigen. Inflammation is caused by a specific defense system response,
When mediated by, or related to, it is said to be the result of responding to a specific defense system. Examples of inflammation as a result of the response of the specific defense system include responses to antigens such as rubella virus, autoimmune diseases, and delayed-type hypersensitivity reactions mediated by T cells (eg, Mantaux) Found in individuals who test "positive"). Chronic inflammatory diseases and rejection of organs such as solid transplants and kidneys, and bone marrow transplants are also examples of the inflammatory response of the specific defense system.

The reaction of the term “non-specific defense system” of the present invention
Refers to a reaction mediated by leukocytes that is incapable of immune memory. Such cells include granulocytes and macrophages. As used herein, inflammation, if caused by or mediated by a response of a non-specific defense system,
Alternatively, when related, it is said to be the result of responding to a non-specific defense system. Examples of inflammation, caused at least in part by the response of the non-specific defense system, include inflammation associated with the following conditions: Adult respiratory distress syndrome (ARDS) or multiple organ injury syndrome resulting from sepsis, trauma, or bleeding; reperfusion injury of myocardium or other tissues; acute glomerulonephritis; reactive arthritis; skin with acute inflammatory components Disease; other central nervous system inflammatory diseases such as acute purulent meningitis or seizures; heat injury; hemodialysis; leukopheresis transfusion; ulcerative colitis; localized ileitis; necrotizing enterocolitis; granular Transfusion-related syndromes; and cytokine-induced toxicity.

As mentioned above, binding of the ICAM-3 molecule to members of the CD-18 family molecule is of central importance for cell adhesion. Through the process of adhesion, leukocytes can continuously monitor the presence of foreign antigens in animals. These processes, although usually desirable, also cause solid organ transplant rejection (eg, kidney), non-solid organ transplant rejection (eg, bone marrow) tissue transplant rejection, and other autoimmune diseases. Therefore, any method capable of weakening or inhibiting cell adhesion is intended for solid organ transplant recipients (particularly kidney transplants), non-solid organ transplant recipients (particularly bone marrow transplants), tissue transplant recipients or autoimmune diseases It is highly desired by patients.

Monoclonal antibodies against members of the CD-18 family inhibit many of the adhesion-dependent functions of leukocytes. This function includes adhesion to endothelial cells (Haskard, D. et al., J. Immu
nol. 137: 2901-2906 (1986)), the same type of adhesion (Rothlei
n, R. et al., J. Exp. Med. 163: 1132-1149 (1986)), antigen and mitogen-induced proliferation of leukocytes (Davignon, D. et al.
Proc. Natl. Acad. Sci., USA 78: 4535-4539 (198
1)), Antibody formation (Fischer, A. et al., J. Immunol. 136: 3198
-3203 (1986)), and, for example, cytotoxic T cells (Krensky, AM et al., J. Immunol. 132: 2180-2182 (198
4)), macrophages (Strassman, G. et al., J. Immunol. 1)
36: 4328-4333 (1986)), and all cells involved in the antibody-dependent cytotoxic response (Kohl, S. et al., J. Immunol.
l.133: 2972-2978 (1984)), including all leukocyte effector functions. In all of the above functions, antibodies inhibit the ability of leukocytes to adhere to the appropriate cell substrate, thereby inhibiting the biological functions involved in binding. Such a function is provided by ICAM-
As long as the 3 / LFA-1 interaction is involved, it can be suppressed by anti-ICAM-3 antibodies.

Therefore, monoclonal antibodies capable of binding to ICAM-3 can be used as anti-inflammatory agents in mammalian specimens. Importantly, such anti-inflammatory agents are unlike common anti-inflammatory agents in that they can selectively inhibit adhesion and have no other side effects such as the nephrotoxicity seen with conventional drugs Different in that.

Such ICAM-3, especially the soluble form, can act in the same manner as antibodies against members of the CD-18 family and can be used to suppress inflammation. In addition, ICAM-
Functional derivatives and antagonists of 3 can also be used to suppress inflammation.

1. Suppressor of Delayed Type Hypersensitivity ICAM-3 mediates, in part, the adhesion events necessary to trigger an inflammatory response, such as delayed type hypersensitivity. Thus, antibodies (particularly monoclonal antibodies) that can bind to ICAM-3 have therapeutic potential to attenuate or eliminate such reactions.

Alternatively, since ICAM-3 is an antagonist of the ICAM-1 / LFA-1 interaction, ICAM-3 (especially a soluble form), or a functional derivative thereof, can be used to suppress delayed hypersensitivity reactions. .

These potential therapeutic uses can be utilized in either of two ways. First, a composition comprising an anti-ICAM-3 antibody or a soluble derivative of ICAM-3 can be administered to a patient who has experienced a delayed type hypersensitivity reaction. For example, such compositions can be provided to an individual who has been contacted with an antigen, such as poison ivy, poison oak, and the like. In another embodiment, a monoclonal antibody capable of binding ICAM-3 is administered to the patient along with the antigen. This is to prevent a subsequent inflammatory response. Thus, administration of the anti-ICAM-3 antibody and the antigen temporarily renders the individual tolerant to the next presentation of the antigen.

2. Treatment of chronic inflammatory diseases Since LAD patients lacking LFA-1 cannot raise inflammatory response, the antagonism of LFA-1 natural ligand
It is thought to inhibit the inflammatory response. Due to the ability of antibodies to ICAM-3 to inhibit inflammation, chronic inflammatory diseases and erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of diabetes, Reynolds syndrome, The basis for therapeutic use in treating autoimmune diseases such as rheumatoid arthritis and the like is established. Such antibodies may also be employed as a therapy for the treatment of psoriasis.

Generally, the anti-ICAM-3 antibodies of the present invention are administered alone or in combination with anti-ICAM-1 and / or anti-ICAM-2 antibodies and are used to treat diseases currently treatable with steroid therapy. Can be done.

According to the present invention, such inflammation and immune rejection response are suppressed by providing a subject in need of such treatment with an anti-inflammatory agent in an amount sufficient to suppress the inflammation ( That is, they can be obstructed or weakened).
Suitable anti-inflammatory agents include an antibody capable of binding to ICAM-3; a fragment of the antibody, which is capable of binding to ICAM-3; a substantially pure ICAM-3; a functional derivative of ICAM-3 Non-immunoglobulin antagonists of ICAM-3 or non-immunoglobulin antagonists of ICAM-3 other than LFA-1. Particularly preferred is ICAM-3
Is an anti-inflammatory agent consisting of a soluble functional derivative of Such anti-inflammatory treatments further include antibodies capable of binding LFA-1; non-immunoglobulin antagonists of LFA-1; substantially pure ICAM-1 and / or ICAM-2, or derivatives thereof, or Administering an agent selected from the group consisting of anti-ICAM-1 antibodies and / or anti-ICAM-2 antibodies or fragments thereof may be included.

The present invention further includes a method for suppressing the inflammatory response of the specific defense system described above, further comprising providing an immunosuppressive agent to the subject. Such immunosuppressants are preferably provided at a lower dose than normally required (ie, a "sub-optimal" dose). The use of a sub-optimal dose is possible because of the synergistic action with the agents of the invention. Suitable immunosuppressants include, but are not limited to, dexamethasone, azathioprine, ICAM-1, ICAM-2, or cyclosporin A.

3. Therapies for Non-Specific Inflammation Many inflammatory responses result from responses of the "non-specific defense system" and are mediated by leukocytes unable to memory. Such cells include granulocytes and macrophages. As used herein, inflammation is said to be the result of a response of a non-specific defense system when caused by, mediated by, or associated with the response of the non-specific defense system. Examples of inflammation, caused at least in part by the response of the non-specific defense system, include inflammation associated with the following conditions:
Adult respiratory distress syndrome (ARDS) or multiple organ injury syndrome resulting from sepsis, trauma, or bleeding; reperfusion injury of myocardium or other tissues; acute glomerulonephritis; reactive arthritis; skin with acute inflammatory components Disease; other central nervous system inflammatory diseases such as acute purulent meningitis or seizures; heat injury; hemodialysis; leukopheresis transfusion; ulcerative colitis; localized ileitis; necrotizing enterocolitis; granular Transfusion-related syndromes; and cytokine-induced toxicity.

The anti-inflammatory agent of the present invention is a compound that can specifically attenuate the interaction between the CD-18 complex on granulocytes and endothelial cells. Such antagonists include ICAM-3; ICAM
-3 functional derivative; ICAM-1, ICAM-2, or CD
Non-immunoglobulin antagonists other than -18 family member molecules are included.

B. Suppressor of Organ and Tissue Rejection ICAM-3, especially the soluble form, can act similarly to antibodies to members of the CD-18 family, thus rejecting organs or tissues caused by any cell adhesion-dependent function Can be used to suppress Furthermore, anti
ICAM-3 antibodies, functional derivatives of ICAM-3, and ICAM-
Three antagonists can also be used to suppress such rejection.

ICAM-3 and anti-ICAM-3 antibodies are useful for rejecting solid organs or tissues, eg, kidneys, in mammalian subjects.
It can be used to prevent rejection of non-solid organs, such as bone marrow, or to weaken the autoimmune response. Importantly, the use of monoclonal antibodies that can recognize ICAM-3 allows organ transplantation even among HLA-incompatible individuals.

C. Addition to the introduction of an antigenic substance administered for therapeutic or diagnostic purposes For example, the immune response to a therapeutic or diagnostic agent such as bovine insulin, interferon, tissue-type plasminogen activator, or murine monoclonal antibody Substantially diminishes the therapeutic or diagnostic value of such agents and, in some cases, causes diseases such as serum sickness. Such situations can be alleviated by using the antibodies of the present invention. In this aspect of the invention, the anti-ICAM-3 antibody is administered in combination with a therapeutic or diagnostic agent. The addition of the antibody prevents the recipient from recognizing the drug, and thus prevents the initiation of an immune response to the drug. The absence of such an immune response allows the patient to further receive the therapeutic or diagnostic agent.

ICAM-3 (especially solubilized form) or a functional derivative thereof may be used alone, or in combination with ICAM-1 or ICAM-2, or in combination with an antibody capable of binding LFA-1 in the treatment of disease. Can be used. Thus, in solubilized form, such molecules can be used to inhibit organ or transplant rejection. ICAM-3,
Alternatively, a functional derivative thereof can be used to reduce the immunogenicity of a therapeutic or diagnostic agent in a manner similar to anti-ICAM-3 antibodies.

D. Suppressors of Tumor Metastasis The agents of the present invention can also be used to inhibit hematopoietic tumor cell metastasis, which requires a functional member of the CD-18 family for migration. In this aspect of the invention,
In a subject requiring such treatment, a sufficient amount of a drug (for example, an antibody capable of binding to ICAM-3;
A toxin conjugate of the antibody; a fragment of the antibody that can bind to ICAM-3; a toxin derivative of the fragment; a substantially pure ICAM-3: ICAM
Functional derivative of ICAM-3; or ICAM-1 or ICAM-
2, non-immunoglobulin antagonists of ICAM-3).

In a further aspect of the present invention there is provided a method for inhibiting the growth of a tumor cell expressing ICAM-3. In particular, the method comprises providing to a subject in need of such treatment a sufficient amount of an agent to inhibit the growth. Suitable agents include an antibody capable of binding to ICAM-3; a toxin derivative of the antibody; a fragment of the antibody that can bind to ICAM-3; a toxin derivative of the fragment; CD-18
Functional derivatives derivatized with toxins of members of the family molecules or members of the CD-18 family molecules are included.

In yet another aspect of the present invention, there is provided a method of inhibiting the growth of LFA-1-expressing tumor cells. In particular, the method comprises providing to a subject in need of such treatment a sufficient amount of an agent to inhibit the growth. Suitable agents include toxin-linked ICAM-3; a functional derivative of ICAM-3 derivatized with a toxin.

E. Control of HIV Infection and Prevention of Transmission of HIV-Infected Cells In yet another aspect of the present invention, there is provided a method for suppressing HIV infection. In particular, the method comprises providing an HIV infected individual with an effective amount of an HIV infection inhibitor. The present invention particularly relates to a method for inhibiting HIV-1 infection, but the method also applies to any HIV mutant (eg, HIV-2, etc.) that can infect cells, as well as can be inhibited by the agents of the invention. It will be understood that it can be applied in the manner described above. Such a mutation is equivalent for the purposes of the present invention.

One of the present invention relates to LFA-stimulated by HIV infection.
1 and in some cases the expression of LFA-1 binding ligand promotes the cell-cell adhesion reaction, which can increase the contact time between infected and uninfected cells,
It is based on the realization that it promotes the transfer of virus from infected cells to uninfected cells. Therefore, LF
Agents of the invention that can modulate A-1 / ligand interactions are H
IV, and in particular infection by HIV-1 can be suppressed. One way that molecules that inhibit the LFA-1 / ligand interaction can suppress HIV infection is that the L-expressed cells expressed by HIV-infected cells
By reducing the ability of the FA-1 ligand to bind to the CD11 / CD18 receptor on healthy T cells. Alternatively, the molecule that inhibits the LFA-1 / ligand interaction is HIV
Healthy T cells of CD11 / CD18 receptor expressed by infected cells
The ability of the cell to bind to LFA-1 can be reduced. To reduce the ability of cells to bind to the CD11a / CD18 receptor or LFA-1 binding ligand, ICAM-3,
ICAM-3 fragments, functional derivatives of ICAM-3, or anti-ICAM-3 antibodies can be used.

The agent of the present invention is provided to a recipient subject in an amount sufficient to achieve suppression of HIV infection. A dose of a drug, such as by administration, is said to be sufficient to “suppress” HIV infection if it is sufficient to attenuate or prevent such HIV infection. The drug is intended to be offered to patients who have been refuted for or have been affected by HIV infection.

The agents of the present invention are for either the "prevention" or "therapeutic" purpose of treating HIV infection. When used for prophylaxis, the drug may be administered before any symptoms of a viral infection (eg, before, at, or shortly after such an infection, but before any symptoms of such an infection have appeared). ) Provided. Prophylactic administration of drugs can prevent or attenuate subsequent HIV infections. When used therapeutically, the agent is provided upon (or shortly after) detection of virus-infected cells. By therapeutic administration of the drug. Further HIV transmission is weakened.

Thus, the agents of the present invention can be administered either before the onset of viral infection (to suppress expected HIV infection) or after the actual detection of such virus-infected cells (to suppress further infection). Can be provided.

In a further aspect, the present invention provides improved therapies for AIDS, as well as enhanced methods for suppressing HIV infection, and particularly HIV-1 infection. This includes administering together:

(I) ICAM-3, soluble ICAM-3 derivative, CD11 (CD11
a, CD11b or CD11c), soluble CD11 derivative, CD1
8, a soluble CD18 derivative, or a CD11 / CD18 heterodimer, or a soluble derivative of the CD11 / CD18 heterodimer and / or (II) an antibody capable of binding to ICAM-3,
And (III) cell or particle-related CD4 or a soluble derivative of CD4 and / or (IV) a molecule (preferably an antibody or antibody fragment) capable of binding to CD4.

In a further aspect of the present invention, there is provided a method for inhibiting the migration of HIV infected cells. Specifically, the method comprises:
Administering an effective amount of an anti-migration agent to an individual infected with HIV.

The anti-migration agent of the present invention contains LFA-
The ability to bind to one ligand can be reduced. ICAM−
3, a fragment of ICAM-3, a functional derivative of ICAM-3, or an anti-ICAM-3 antibody. An antibody that binds to ICAM-3 is CD1 of ICAM-3 expressed by HIV-infected T cells.
Suppresses migration by reducing the ability to bind to cells that express the 1 / CD18 receptor. Antibodies that can bind to ICAM-3 can be used to reduce the ability of the cells to bind to the CD11 / CD18 receptor.

The agent of the present invention is provided to the recipient subject in an amount sufficient to inhibit the migration of T cells infected with HIV (or other virus). A dose of an agent, such as by administration, is said to be sufficient to “suppress” T cell migration if it is sufficient to weaken or prevent such migration.

Such compound (s) are referred to as “preventative”
Alternatively, it may be for any of the purposes of "treatment".
When used for prophylaxis, the agents of the present invention may be administered prior to any symptoms of a viral infection (eg, before, at, or immediately after such infection,
Provided before all symptoms of such an infection have appeared). Prophylactic administration of the agent can prevent or attenuate subsequent migration of virus-infected T cells. When used therapeutically, the agent is provided upon (or shortly after) detection of virus-infected T cells. Therapeutic administration of the drug reduces such T cells from migrating further.

Thus, the agents of the present invention can be provided either before the onset of viral infection (to suppress the expected migration of infected T cells) or after the actual detection of such virus infected cells.

F. Treatment of Asthma In a further aspect of the invention, an agent capable of modulating the LFA-1 / ICAM-3 interaction is used for the treatment of asthma. In particular, the method comprises administering to an individual in need of such treatment an effective amount of an anti-asthmatic agent.

The anti-asthmatic agent of the present invention includes ICAM-3, a fragment of ICAM-3, a functional derivative of ICAM-3, or an anti-ICAM, which can impair the ability of cells to bind to LFA-1.
-3 antibody. Antibodies that bind to ICAM-3 inhibit eosinophil migration by impairing the ability of ICAM-3 expressed on these cells to bind to cells expressing the CD11 / CD18 receptor.

The anti-asthmatic agent of the present invention is provided to a recipient subject in an amount sufficient to reduce or attenuate the severity, severity, or duration of asthma symptoms.

The agents of the present invention can be administered alone or in combination with one or more anti-asthmatic agents. Such anti-asthmatics include, for example, methylxanthine (such as theophylline), beta-adrenergic agonists (such as catecholamine, resorcinol, saligenin, and ephedrine), glucocorticoids (such as hydrocortisone), chromones (such as cromolyn sodium), And anticholinergics (such as atropine), which in combination with the agents of the present invention, reduce the amount of such agents needed to treat asthma symptoms.

The agents of the present invention are for either "preventive" or "therapeutic" purposes. When used for prophylaxis, the drug is provided before any symptoms of asthma appear. Prophylactic administration of the drug can prevent or attenuate the subsequent asthmatic response. When used for treatment,
The drug is provided at the onset of (or shortly after) the symptoms of asthma. Therapeutic administration of the drug attenuates substantial asthma symptoms. Therefore, the drug of the present invention
It may be provided either before the onset of the expected asthma attack (to reduce the pain, time or extent of the expected asthma attack) or after the onset of the attack.

G. Diagnostic and Prognostic Applications Monoclonal antibodies capable of binding to ICAM-3 can be used in methods for imaging or visualizing ICAM-3 expression sites and sites of inflammation in patients. In such uses, the anti-ICAM-3 antibody is detectably labeled by use of a radioisotope, an affinity label (biotin, avidin, etc.), a fluorescent label, or a paramagnetic atom. Methods for making such labels are well known in the art. The labeled antibody can then be used for imaging for diagnosis. For clinical applications in imaging for the diagnosis of antibodies, see Grossman, HB, Urol. Clin.
Amer. 13: 465-474 (1986), Unger, EC et al., Invest. Radi.
ol. 20: 693-700 (1985), and Khaw, BA et al., Science.
209: 295-297 (1980).

The presence of ICAM-3 expression can also be detected by using a hybridization probe. Such probes include ICAM-3 present in ICAM-3 expressing cells.
MRN capable of binding to a gene sequence or ICAM-3 mRNA sequence
A, cDNA, chromosomal DNA, or synthetic oligonucleotide probes. Techniques for performing such hybridization assays are described in Maniatis, T. et al.
Et al., Molecular Cloning, a Laboratory Manual, Coldspri
ng Harbor, NY (1982), and Haymes, BD et al., Nucleic.
Acid Hybridization, a Practical Approach, IRL Pres
s, Washington, DC (1985), which are incorporated herein by reference.

Detection of the location of ICAM-3 expression by using a labeled antibody or nucleic acid probe can be used to diagnose tumor development. In one diagnostic embodiment, a tissue or blood sample is removed from a subject and incubated in the presence of an already labeled antibody or an antibody that can be labeled therefrom. In a preferred embodiment,
This technique is performed in a non-submerged manner through the use of magnetic imaging, fluorography. Such diagnostic tests can be used, for example, to monitor an organ transplant patient, such as the kidney, for early signs of possible tissue rejection. Such assays can also be performed to determine an individual's susceptibility to rheumatoid arthritis, or other chronic inflammatory diseases.

For example, radiolabeling an anti-ICAM-3 antibody or antibody fragment allows for the detection of the antigen by use of a radioimmunoassay. For Radio Immunoassay (RIA), see Laboratory Techniqu
es and Biochemistry in Molecular Biology, (Work, T.
S. et al., North Holl and Publishing Company, NY
(1978)) “An Introduction to Radioimmune Assay
and Related Techniques "(by Chard, T.), which is incorporated herein by reference. Alternatively, antibodies may be fluorescent compounds, enzymes, or antibodies known in the art. It may be labeled by some other suitable label.

Apart from locating the site of inflammation, antibodies that can bind to ICAM-3 can be used to assay circulating ICAMs in biological fluids using solvents commonly employed in liquid assays.
-3 can be used to check for the presence of. The presence of circulating ICAM-3 in the fluid sample may indicate an inflammatory response or other ICAM-
3 is a faint sign of mediated biological function. Alternatively, the presence of ICAM-3 in amniotic fluid has been associated with the product of complications during pregnancy. All biological fluids can be used for the assay. However, preferred biological fluids are blood, serum, plasma, synovial fluid, amniotic fluid, spinal fluid, or urine.

VIII. Administration of the Compositions of the Invention The therapeutic effect of ICAM-3 is obtained by administering to a patient either the full-length ICAM-3 molecule, or a therapeutically active peptide fragment thereof. Of particular interest are soluble therapeutically active peptide fragments of ICAM-3.

ICAM-3 and its functional derivatives can be obtained synthetically, by recombinant DNA technology, by proteolysis, or by a combination of such methods. Therapeutic treatment of ICAM-3 by the use of a functional derivative of ICAM-3 having an amino acid residue added to enhance binding to the carrier or an amino acid residue added to increase the activity of ICAM-3. The above advantages increase. The scope of the present invention further includes an ICAM-containing amino acid residue that is deleted or includes an alternative amino acid residue as long as it has or acts on the biological or pharmacological activity of ICAM-3. Also included are three functional derivatives.

Antibodies and ICs of the invention disclosed in this specification
Both AM-3 molecules are "substantially free of natural contaminants" when the preparations containing those antibodies and molecules are substantially free of materials as normally and naturally found.
It is said.

The present invention includes (polyclonal or monoclonal) antibodies, active fragments thereof, which are capable of binding to ICAM-3. Such antibodies can be produced either by animal, tissue culture, or recombinant DNA methods.

Administration of ICAM-3, or a molecule derived from ICAM-3, can be performed alone or in combination with location and or ICAM-2. Anti-ICAM-3 antibody or ICAM-
3 or other molecules that can bind to molecules derived from ICAM-3 can be administered alone or in combination with anti-ICAM-1 and / or anti-ICAM-2 antibodies. IC to the patient
When providing an antibody or antibody fragment capable of binding to AM-3, or when providing ICAM-3 (or a fragment, variant, or derivative thereof) to a recipient patient, the dose of the administered drug depends on the patient. Depends on factors such as age, weight, height, gender, general symptoms, and history of living. Generally, it is desirable to provide the recipient with a dose of the antibody in the range of about 1 pg / kg to 10 mg / kg (body weight of the patient), although lower or higher doses may be administered. When providing an ICAM-3 molecule, or a functional derivative thereof, to a patient, it is also desirable that such a molecule be administered at a dose ranging from about 1 pg / kg to 10 mg / kg (the patient's body weight). , Lower or higher doses may be administered. As shown below,
The therapeutically effective dose is that the anti-ICAM-3 antibody will
-1 antibody, anti-ICAM-1 antibody and / or anti-ICAM-
When administered with two antibodies, it can be lower.
As used herein, one compound is said to be further administered together with a second compound if the two compounds are administered in such a close time that they are simultaneously found in the serum of the patient.

Both antibodies that can bind to ICAM-3, and ICAM-3 itself, are administered intravenously, intramuscularly, subcutaneously, intestines,
It can be administered topically or parenterally. Where the antibody or ICAM-3 is administered by infusion, the administration can be by continuous infusion, or in single or multiple units.

The agents of the present invention are provided to a recipient subject in an amount sufficient to be "physiologically effective." An amount is said to be physiologically effective if the dose of the agent, such as by administration, is sufficient to diminish or counteract the physiological effects associated with ICAM-3. For example, one agent of the invention that is provided to a patient for the purpose of suppressing inflammation may be physiologically effective when provided at a dose sufficient to “suppress” inflammation.

In addition, the anti-ICAM-3 antibody, or fragment thereof, can be administered alone or in combination with one or more other immunosuppressive agents, particularly for organ or tissue transplant recipients. . Administration of such compound (s) may be for either "prophylactic" or "therapeutic" purpose. When used for prophylaxis, the immunosuppressive compound is provided before any inflammatory response or symptoms appear (eg, before, at, or shortly after, organ or tissue transplantation, but without any symptoms of organ rejection). Before it comes out). Prophylactic administration of the compound (s) can prevent or attenuate subsequent inflammatory responses (eg, rejection of transplanted organs or tissues). When used for therapy, the compound (s) are provided at the onset of (or shortly after) the actual inflammatory condition. Therapeutic administration of the compound (s) attenuates the actual inflammation (eg, rejection of a solid organ such as a transplanted kidney or a non-solid organ such as tissue or bone marrow).

Thus, the anti-inflammatory agents of the present invention may be provided either before the onset of inflammation (to suppress expected inflammation) or after the onset of inflammation.

If administered and tolerated by the recipient patient,
The composition is referred to as "pharmaceutically acceptable." Such an agent is said to be administered a "pharmaceutically effective amount" if the amount administered is physiologically sufficient. An agent is physiologically sufficient if its presence results in a detectable change in the physiology of the recipient patient.

The antibodies and ICAM-3 molecules of the invention can be formulated in a known manner for preparing pharmaceutically useful compositions. In these methods, these materials, or functional derivatives thereof, are combined with a pharmaceutically acceptable carrier excipient.
Suitable excipients and their formulations include, for example, other human proteins, such as human serum albumin, which are described in Remington's Pharmaceutical Sciences.
(16th edition, edited by Osol, A., Mack, Easton PA (1980)). Such compounds comprise an effective amount of an agent of the present invention and a suitable amount of carrier to form a pharmaceutically acceptable composition suitable for effective administration. further,
The antibodies of the present invention can be humanized by chimerization or CDR grafting, making them more "pharmaceutically acceptable" to a patient. Methods for chimerizing antibodies, particularly anti-ICAM-1 antibodies, are described in other specifications and the like. This is the UK patent application No. 90009548.0, No. 90009549.8,
And PCT Application Nos. PCT / US91 / 02942 and PCT / US91 / 0294 filed on April 27, 1991 with the United States Receiving Office.
No. 6, which are incorporated herein by reference.

Yet another pharmaceutical method can be used to control the duration of action. Controlled release preparations may be made by the use of polymers to synthesize or absorb the agents of the present invention.
The rate and time of controlled delivery can be controlled to some extent by varying the concentration and method of introduction of the macromolecules by selecting an appropriate macromolecular matrix. Another way to control the time of controlled delivery is
The incorporation of the agents of the present invention into particles of a polymeric material such as a polyester, polyamino acid, hydrogel, poly (lactic acid) or ethylene vinyl acetate copolymer. Alternatively, instead of introducing these drugs into the polymer particles, these materials can be encapsulated in microcapsules, colloid drug delivery systems, or microemulsions. Among them, the microcapsules are prepared by, for example, coacervation, interfacial polymerization, gelatin or poly (methylmethacrylate) microencapsulation. And
Colloidal drug delivery systems include, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules.

The invention further encompasses a pharmaceutical composition comprising: (a) an anti-inflammatory agent (an antibody capable of binding to ICAM-3; ICAM
Fragment of the antibody capable of binding to ICAM-3; ICAM-3; ICAM
Functional derivatives of ICAM-1 or ICAM-1 and ICAM-2
(B) at least one immunosuppressive agent. Suitable immunosuppressants include dexamethasone, azathioprine and cyclosporin A.

Having now described the invention in general, referring to the following examples, one can more readily understand the agents and the methods of obtaining them. The following examples are provided for illustrative purposes, and unless otherwise indicated,
It does not limit the invention.

Example 1 A new adhesion ligand to LFA-1, ICAM
Attachment of the characterized cell line and lymphocytes to LFA-1 coated plates was performed as previously described [Dustin et al., Cold Spring Harbor Symp. Quan.
t. Biol. 54: 753-765 (1989)]. JY lysate (solubility)
LFA-1 purified from a well was placed in a 96-well microtiter plate (Linbro-titertek) at 1100 sites / site.
Adsorbed at m ² . To block non-specific binding sites with 1% BSA, washed with _{PBS / 5% FBS / 2mMMgCl 2} /0.5%HSA ( assay medium). Specific inhibition of LFA-1 was achieved by combining microtiter wells with 1/200 diluted TS1 / 22 (anti-LFA-1α) ascites.
Incubation was performed at RT for 30 minutes. Resting T-cells were isolated by plastic deherance and nylon wool filtration and were 91% CD2 ⁺ . On the other hand, PHA-blasts were generated by culturing the cells in the presence of 10 μg / ml phytohemagglutinin (PHA). Fluorescent dye B
Labeled with CECF (Molecular Probes Inc.), washed and resuspended in assay medium. MAb pretreatment of cells was performed by incubating with 1/200 diluted ascites at 4 ° C. for 45 minutes, then transferring 10 ⁵ cells to each well. Solid phase LFA-
Let the cell line adhere to 1 for 1 hour at 37 ° C and remove
Individual 23 gauge, needle aspirated, while lymphocytes were centrifuged at 30 × g for 5 minutes and incubated at 37 ° C. for 30 minutes. Unbound lymphocytes should be removed 100 times between each wash.
８ 8 times, and by flicking the medium,
Removed from plate. Flicking is more effective to completely remove unbound T cells that are more difficult to remove due to their small size. Fluorescence was quantified directly from a 96-well plate by a fluorescence intensity (concentration) analyzer (Baxte). Using all MAbs at saturating concentration (1/200 diluted ascites), TS1 / 22 (anti-CD11a, IgG1) [Sanchez-Madrid et al. Proc. N
atl. Acad. Sci. USA 79: 7489-7493 (1982)], RR1 / 1 (anti-
ICAM-1, IgG1) [Rothlein et al.
J. Immunol. 137: 1270-1274 (1986), CBR-IC2 / 2 (anti-ICA
M-2, IgG2a) [de Fougerolles et a
l.) J. Exp. Med. Submitted (1991) (in press)], and CBR-IC3 / 1 (anti-ICAM-3, IgG1). CB
R-IC3 / 1 is Balb / c mouse injected with murine myeloma P3X63Ag8.653 and SKW3 [Gefter et al.
Gen. 3: 231-236 (1977)], and 600 hybridomas were purified from SKW3 purified LFA-
Screened for their ability to inhibit binding to 1. CRB-IC3 / 1 was purified IFA-1, ICAM-1
It did not react with COS cells transfected with either 2 or LFA-1 cDNAs (data not shown). One of four representative experiments is shown, with errors indicating one standard deviation.

Example 2 Cellular ICAM-1, ICAM-2, and ICAM-3
Flow cytometric analysis of expression All cell lines used were as previously described [Hibbs et al. Clin. Invest. 85: 674-681 (1992).
0)]. Dextran sedimentation of peripheral blood mononuclear cells (PBMC) and F
[Dustin et al. (Du
stin, et al.) J. Cell Biol. 107: 321-331 (1988)].
Neutrophils were recovered from the cell pellet and mixed red blood cells were removed by hypotonic lysis. Lymphocytes and monocytes were separated by cytometry analysis using forward and vertical light scatter and confirmed with monocyte and T cell specific MAbs. PBMC
Using plastic adherence to enrich for lymphocytes obtained from, cells were harvested at 10 μg / ml phytohemagglutinin (PH
The cells were cultured in the presence of A). The samples were analyzed using an EPICS V flow cytometer and EPICSI mmune-Brite fluorescent beads (Co
The fluorescence was quantified using the following method. ICA in resting lymphocytes
The expression of M-3 is 2- to more than either CD3 or LFA-1.
Monocyte expression was 3-fold greater with ICAM-3 than LFA-1 with 3-fold greater. ICAM-3 expression levels in neutrophils were comparable to those of Mac-1 (CD11b / CD18). Treatment of the cells with phosphorylase C revealed no PI-linked ICAM-3.

Example 3 Immunoprecipitation of ICAM-3 Cells were labeled with ¹²⁵ I using iodogen [Kishimoto et al., J. Biol. Chem. 264: 3588-3595.
(1989)]. Cells were lysed with Triton X-100 (1%). The lysate was pre-cleared on Sepharose conjugated to bovine-IgG and then incubated with the appropriate MAb-conjugated Sepharose for 2 hours. Beads 50 mM Tri
s Washed and boiled in sample buffer containing 1% SDS and 1% 2-mercaptoethanol. Non-reduced samples were boiled in sample buffer without 2-mercaptoethanol and treated with 20 mM iodoacetic acid. Samples were analyzed on a 7% vertical slab (separation plate) polyacrylamide gel as previously described [Laemml
i, UK) Nature 227: 680-685 (1970)], and the proteins were observed by autoradiography. Samples were prepared as previously described [Tarentino et al. Biochemistry 2].
4: 4665-4671 (1985)] using N-Glycanase (Genzy).
me) and cleavage of all N-linked oligosaccharides of the peptide backbone was performed at a concentration (10 units / ml, 37 ° C, 18 ° C).
Time) was determined to be optimal. MHC
Class I contains on N-linked carbohydrates, but ICAM-2
(M _r 60,000, 6N-binding site, M _r 28,383 backbone)
Was highly glycosylated.

Example 4 Distribution of ICAM-3 To further examine the tissue distribution of ICAM-3 protein by flow cytometry, the anti-ICAM-3 antibody of the present invention was used. Table 3 summarizes cell types showing surface expression of ICAM-3. ICAM-3 expression appears to be restricted to hematopoietic cells. Flow cytometry data was confirmed by immunohistochemical staining of tissue sections using labeled anti-ICAM-3 antibody (data not shown). As with flow cytometry data, ICAM-3 was also used in immunohistochemical studies.
It was found that expression appeared to be restricted to hematopoietic cells.

Example 5 Purification of ICAM-3 Anti-ICAM immobilized on a matrix by methods known in the art
ICAM-3 was purified by immunoaffinity chromatography using the -3 antibody CBR-IC3 / 1. Many sources were used as starting materials for the isolation of ICAM-3. These include, but are not limited to, SKW3, human thymoma cell lines, and human tonsils. The method used for the purification of ICAM-3 was essentially that described in the literature [de Fougerol et al.
rolles et al.) J. Exp. Med. 175: 185-190 (1992)]. Those skilled in the art will appreciate that the washing and elution regimes and reagents used for immunoaffinity chromatography will vary slightly for each antigen to be purified and each antibody used for immunoaffinity chromatography.

As described below, ICAM-3 purified from SKW3 cells or human tonsils has a molecular weight of about 120-124 kD, whereas ICAM-3 purified from neutrophil-derived lysates has a molecular weight of about 120-124 kD.
Gave a wide band of 150kD.

ICAM-3 purified in this manner appeared to retain its ability to bind its anti-ICAM-3 antibody and LFA-1 on various cells (see FIGS. 6 and 7).

Example 6 Identification of ICAM-3 of Various Molecular Weights Immunoprecipitation and analysis by polyacrylamide gel electrophoresis were used to determine the molecular weight of ICAM-3 on various cell types. It was found that the molecular weight of ICAM-3 immunoprecipitated from neutrophils was different from that of immunoprecipitated from lymphocytes. ICAM-3 isolated from lymphocytes and lymphoid cell lines appeared as a band with a molecular weight of approximately 120-124 kD. The molecular weight of ICAM-3 immunoprecipitated from neutrophils is
Slightly larger than that expressed in lymphoid cells, about 120
From 150 to 150 kD (Figure 4).

Such a change in size is a glycoprotein
It is not unusual for members of the ICAM family. ICAM-1 shows similar changes in molecular weight between different cell types. It was shown that the change in molecular weight observed in ICAM-1 was caused by a change in the degree of glycosylation. Therefore,
The most likely cause of a change in the molecular weight of ICAM-3 is a difference in glycosylation. One of skill in the art can readily confirm this by subjecting ICAM-3 isolated from neutrophils to various glycosidases and other enzymatic treatments and observing the effect on molecular weight. .

The change in the degree of glycosylation of ICAM-1 was MAC-1 (CD1
1b / CD18) was shown to affect binding. Therefore, ICA
Altering the degree of glycosylation of M-3 can produce ICAM-3 derivatives with altered binding affinity for various ligands of ICAM-3, such as members of the glycoprotein CD11b / CD18s. It is possible.

Example 7 Mice were injected with ICAM-3 antibody adjuvant and ICAM-3 protein purified from SKW3 or tonsil cells as described above.
Monoclonal antibodies were generated from the immunized animals using methods known in the art.

Table 4 summarizes the obtained various anti-ICAM-3 antibodies. Various antibodies were identified by ELISA based on their ability to react with the purified immobilized ICAM-3. Next, ICAM-3
Positive mA on various cell lines known to be positive
b was screened and immunoprecipitated from radiolabeled ICAM-3-positive cell lysates. mAbs were purified by PMA-induced SKW in the presence of anti-ICAM-1 and anti-ICAM-2 antibodies.
Three cells were tested for their ability to inhibit aggregation. Alternatively, anti-ICAM-3 antibodies can be identified by their ability to inhibit the binding of SKW3 cells to immobilized purified ICAM-3.

One piece of evidence indicating the presence of a third LFA-1 ligand is the presence of a pathway for PMA-stimulated SKW3 cell aggregation, independent of ICAM-1 / ICAM-22. We were able to inhibit this aggregation with either mouse anti-ICAM-3 polyclonal antiserum or a combination of CBR-IC3 / 1 and CBR-IC3 / 2.

Example 8 Immunoassay using anti-ICAM-3 antibody Anti-ICAM-1 antibody (RR1 / 1), anti-ICAM-2 antibody (CBR-IC)
2/2) and anti-ICAM-3 antibodies (CBR-IC3 / 1 and CBR-IC
3/2 combination), (1) Purification of SKW3 cells by PMA stimulation
Adhesion to ICAMs, (2) phytohemagglutinin (PHA)
The cells were tested for cell division upon stimulation and (3) their ability to inhibit mixed lymphocyte response (MLR).

Already, PMA activates LFA-1 and thus its ICAM-1
Has been shown to be able to increase the ability to bind to [Dustin et al. Nature 341: 619 (1989)]. FIG. 5 demonstrates that a similar activation mechanism exists for LFA-1 / ICAM-3 binding.

Purified ICAM- of SKW3 cells stimulated by PMA with various antibodies
1 and the ability to inhibit binding to ICAM-3 was tested. FIG.
The CBR-IC3 / 1 and CBR-IC3 / 2 antibodies, when present individually, do not effectively inhibit the attachment of PMA-stimulated SKW3 cells to purified ICAM-3. However, the use of a combination of these two antibodies inhibits attachment of SKW3 cells to ICAM-3 under PMA stimulation. De Fougerolles e
As described by T. al., J. Exp. Med.], the CBR-IC3 / 1 monoclonal antibody is itself an ICAM-antibody to purified LFA-1.
3 can be inhibited.

Further analysis was performed to determine whether the binding of PMA-stimulated SKW3 cells to ICAM-3 was temperature- and cation-dependent. Like ICAM-1, ICAM-3 /
LFA-1 binding was found to be temperature dependent (see FIG. 8) and cation dependent (data not shown).

FIG. 9 shows the effect of various antibodies on PHA-stimulated cell division. As already mentioned, [Krensky et al.
l.) J. Immunol. 131: 611-616 (1983)], PHA is anti-ICAM
Stimulating cell proliferation in such a way that it can be inhibited by anti-LFA-1 in anti-ICAM-1 antibody or anti-ICAM-2 antibody, or
The combination of 1 and anti-ICAM-2 antibody had little effect on PHA-stimulated cell division. However, 1) Anti-IC
AM-1 antibody, anti-ICAM-2 antibody and anti-ICAM-3 antibody,
2) Two anti-ICAM-3 antibodies (IC3 / 1 and IC3 / 2) and 3) a combination of anti-ICAM-1 and anti-ICAM-3 antibodies were effective for PHA-stimulated cell division.

Example 9 Mixed Lymphocyte Reaction The MLR test is used to assess immunological reactivity. The assay basically consists of exogenous chemically fixed cells (stimulator cells) from different solids.
Including the solution containing PELs [responder cells] and measuring the level of response as an index of the proliferation of responding cells [Krensky et al. J. Immunol. 131: 611.
-616 (1983)]. This assay is the first step in identifying components useful for treating transplant rejection.

The effects of various antibodies and combinations of antibodies on the MLR reaction are shown in FIG. In summary, anti-ICAM-3 alone has a low level of effect. However, anti-ICAM-1 antibody and anti-ICAM-
In combination with 3 antibodies, it is seen that MLR inhibition produces a high level of effect. The highest response was obtained with a combination of anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3 antibodies.

Example 10 Peptide sequence of ICAM-3 ICAM-3 was purified from human tonsils by immunoaffinity chromatography according to the method described in Example 5. Purified ICAM-3 was enzymatically digested with the Lys-C enzyme by known methods, and peptide fragments were separated by HPLC. FIG. 11 shows the various protein peaks on a gas chromatograph that were observed during normal digestion. Peaks 10 and 17 were identified as containing peptide fragments of sufficient size and structure to be sequenced (hereinafter, referred to as
NK-10 and NK-17 peptides, respectively).

The amino acid sequences of the NK-10 and NK-17 peptides were determined by standard techniques. The sequence of the NK-10 protein was found to be KIDRATCPQHLK (SEQ ID NO: 1).
The presence of the first lysine (K) residue in the sequence was deduced from the fact that Lys-C cleaves the protein after the lysine residue.

The sequence of the NK-17 peptide was found to be KIALETSLSK (SEQ ID NO: 2). Like the NK-10 protein, the first lysine residue was deduced and confirmed by DNA sequencing.

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

Example 11 cDNA Cloning of ICAM-3 Degraded oligonucleotide probes were prepared based on the peptide sequences obtained above. In addition, the probes were designed to include the codon preferences found in the ICAM-1 and ICAM-2 nucleotide sequences identified above. NK-17
The nucleotide sequences of the probes based on the amino acid sequences of the protein and NK-10 protein are as follows.

As described above, a cDNA expression library was generated from tonsil RNA [Wong et al., Proc. Natl. Acad.
Sci. USA 82: 7711-7716 (1985)]. The library was screened with the NK-17 probe, and plaques showing positive hybridization were screened again with the NK-10 probe. One clone, clone 11.2,
Was found to have an insert approximately 1.6 to 1.8 kb in length. A diagram showing the location of clone 11.2 and the NK-10 and NK-17 peptides is shown in FIG.

The ends of the insert were sequenced using primers derived from the adjacent vector, and the NK-10 probe was used for internal sequencing. The sequences obtained so far have been read on only one strand. One skilled in the art will readily appreciate that the effectiveness of such sequences can be ascertained by routine experimentation.

The sequences obtained are summarized in FIG. There are a few things to note about this arrangement. First, a sequence highly homologous to the transmembrane region of ICAM-1 is identified. Therefore, ICAM-
If one wishes to produce a soluble fragment of 3, the entire sequence behind the start of the transmembrane region shown in FIG. 12 is deleted. Second,
As can be inferred from the sequence homology present between ICAM-1, ICAM-2 and ICAM-3, the sequence obtained from the 5 'end of clone 11.2 is close to the 5' end of the naturally occurring gene It seems.

Example 12 Sequence Homology Between Various ICAM Molecules The nucleotide and amino acid sequences of the NK-10 and NK-17 peptides, and the nucleotide sequences obtained above were compared to known ICAM-1 and ICAM-2 sequences ( FIG.
-17).

The results suggest a high level of homology between ICAM-3 and both ICAM-1 and ICAM-2. ICAM-1 and ICAM
Sequence homology with -3 is found in the first (FIG. 16) and fourth / fifth domains of ICAM-1, as well as in the transmembrane and several cytoplasmic domains of ICAM-1 (FIGS. 13 and 14). .
Furthermore, when searching the gene bank database,
No other sequences identical or similar to those obtained for ICAM-3 were found. The first of ICAM-3 and ICAM-2
Significant homology was also found between the domains (FIG. 17).

The library was further screened using clone 11.2. Additional clones were found to obtain a cDNA insert of approximately 2-2.4 kD. Since larger sizes have not been identified so far, they may represent full-length cDNA clones.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI G01N 33/577 C12N 15/00 ZNAA (72) Inventor Springer, Timothy A. 02167, Massachusetts, USA, Chestnut Hill, Monadnock Law No. 28 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/09 C07K 16/18 C12Q 1/68 G01N 33/53 G01N 33/569 G01N 33/577 BIOSIS (DIALOG) MEDLINE (STN ) GenBank / EMBL / DDBJ / GeneSeq

Claims (10)

(57) [Claims] 1. A recombinant DNA molecule comprising the amino acid sequence of SEQ ID NOs: 2, 4 and 6, and encoding ICAM-3 capable of binding to LFA-1. 2. The recombinant DNA molecule according to claim 1, comprising the nucleotide sequence according to SEQ ID NOs: 1, 3 and 5. 3. The recombinant DNA molecule according to claim 1, which is capable of expressing ICAM-3 in a cell. 4. A method for detecting the presence of a cell expressing ICAM-3, comprising: (a) incubating the cell or the cell extract in the presence of a nucleic acid molecule capable of hybridizing with ICAM-3 mRNA. And (b) determining whether the nucleic acid molecule has hybridized to a complementary nucleic acid molecule present in the cell or an extract of the cell. 5. ICAM-3 in a liquid sample obtained from a mammalian subject.
Comprising: (a) incubating a biological sample of the subject with a composition comprising an antibody or a fragment of the antibody capable of binding ICAM-3; and (b) A method comprising detecting an antibody bound to a liquid. 6. The method according to claim 5, wherein said liquid is selected from the group consisting of blood, serum, plasma, synovial fluid, amniotic fluid, cerebrospinal fluid or urine. 7. A method for identifying a substance capable of regulating the binding of ICAM-3 to LFA-1, comprising contacting ICAM-3 with LFA-1 in the presence of said substance, Measuring the regulation on LFA-1 binding. 8. The method of claim 7, wherein said LFA-1 and said ICAM-3 are purified, free of naturally occurring impurities. 9. The method according to claim 8, wherein the LFA-1 is expressed on a cell surface,
8. The method of claim 7, wherein said ICAM-3 is purified, free of naturally occurring impurities. 10. The method of claim 10, wherein said ICAM-3 is expressed on the cell surface and said LFA-1 contains no naturally occurring impurities.
The method according to claim 7, which is purified.