HU217176B - Novel intercellular adhesion molecule and its binding ligands, compositions containing thereof and process for producing thereof - Google Patents

Abstract

The present invention relates to an intercellular adhesive mucosa of ICAM-3, which participates in the mainstream on which algal stools recognize and migrate to their site and attach to acellular fibrous substrate. The invention encompasses the aforementioned molecules, assays for their aberration, DNA molecules encoding such a molecule, antibodies that bind such a molecule, and therapeutic and diagnostic compositions comprising such a molecule or its binding antibodies. The invention further extends to the production of the above. ŕ

Description

The present invention relates to a novel intercellular adhesion molecule, designated IC'AM-3, which is involved in the process of recognizing and adhering to a cellular substrate by a population of leukocytes. ICAM-3 mediates cellular interactions with other lymphocytes, macrophages and neutrophils at sites of inflammation and immune response.

The invention relates to compositions comprising ICAM-3 alone or in combination with ICAM-1 and / or ICAM-2, which inhibit intercellular adhesion of granulocyte, lymphocyte or macrophage cells. Such molecules allow the treatment of both specific and non-specific inflammation.

The ICAM-3 of the present invention may be used in the therapeutic or prophylactic treatment of HIV-leukocytes, in particular HIV-1, in patients infected with or exposed to HIV-1 by administering ICAM-3 alone or ICAM-1. and / or in combination with ICAM-2. This makes it possible to treat diseases caused by the HIV virus, such as AIDS (Acquired Immune Deficiency Syndrome).

It can also be used in a therapeutic method for inhibiting the migration of HIV-1 infected cells into the circulatory system to which ICAM-3 is added alone or in combination with ICAM-1 and / or ICAM-2. This allows treatment of diseases caused by the HIV-1 virus, such as AIDS.

It can also be used in a therapeutic method that allows inhibition of T cell death and "syncytia" in HIV-infected individuals to which ICAM-3 is administered alone or in combination with ICAM-1 and / or ICAM-2. This allows treatment of diseases caused by the HIV-1 virus, such as AIDS.

ICAM-3 of the present invention may also be used alone or in combination with ICAM-1 and / or ICAM-2 for the treatment of asthma. The invention further encompasses molecules capable of binding ICAM-3 (hereinafter referred to as anti-ICAM-3). Binding of anti-ICAM-3 to ICAM-3 modulates the biological functions associated with ICAM-3. The binding molecule of the invention may be an antibody, peptide or carbohydrate capable of binding ICAM-3. Such molecules are useful for modifying the biological functions of ICAM-3.

The invention further provides a composition comprising the anti-ICAM-3 binding molecule, alone or in combination with an anti-ICAM-1 and / or antiICAM-2 molecule, which can be used to inhibit intercellular adhesion of granulocytes, lymphocytes or macrophages. The use of such molecules enables the treatment of both specific and non-specific inflammation.

The ICAM-3 of the present invention may also be used in therapeutic and prophylactic methods which allow the inhibition of HIV infection of leukocytes, in particular HIV-1, by administering anti-ICAM-3 alone or anti-HIV to a patient infected with or exposed to HIV-1. In combination with ICAM-1 and / or anti-ICAM-2. This allows treatment of HIV-related diseases, such as AIDS.

The ICAM-3 of the present invention may further be used in a therapeutic method that permits inhibition of HIV-1-infected cell migration within the circulatory system to which an anti-ICAM-3 agent is added alone or in combination with an anti-ICAM-1 and / or antiICAM-2 molecule. . This allows the treatment of diseases caused by HIV-1, such as AIDS.

The anti-ICAM-3 of the invention may also be used alone or in combination with an anti-ICAM-1 and / or antiICAM-2 molecule for the treatment of asthma.

Leukocytes attach to various cellular substrates to protect the body against foreign pathogens such as bacteria or viruses [Eisen Η. V: Microbiology, 3rd edition, Harper and Row, Philadelphia, PA, USA 290-295 and 381-418 (1980). Leukocytes attach to endothelial cells in order to move from the circulatory system to the site of inflammation. In addition, they bind to antigen-producing cells and thus elicit a normal specific immune response. Finally, they bind to appropriate target cells and thus trigger the destruction of a virally infected or tumorous cell.

Recent research on the surface of leukocytes by hybridoma technology has identified molecules that are involved in the functions mediated by the above binding. Briefly, monoclonal antibodies against human T cells [Davignon, D., et al., Proc. Natl. Acad. Sci. USA 78: 4535-4539 (1981)] and a monoclonal antibody against mouse spleen cells (Springer T. et al., Eur. J. Immunol. 9: 301-306 (1979) which bind to the surface of leukocytes and inhibit the above-related binding functions [Springer T. et al., Fed. Proc. 44, 2660-2663 (1985)]. The molecules identified by such antibodies are labeled with Mac-1 and LFA-1 (antigen related to lymphocyte function). Mac-1 is a heterodimeric molecule found on macrophages, granulocytes and large granulation lymphocytes. LFA-1 is a heterodimer molecule found on most lymphocytes [Springer T. A. et al., Immunol. Port. 68: 111-135 (1982)]. These two molecules, together with a third molecule, pl50.95, which exhibits tissue distribution similar to Mac-1, play an important role in cellular adhesion (Keizer, G. et al., Eur. J. Immunol. 15: 1142-1147 (1985).

These leukocyte molecules belong to the family of glycoproteins [Sanches-Madrid F. et al., J. Exper. Med. 158: 1785-1803 (1983); Keizer, G.D., et al., Eur. J. Immunol. 15, 1142-1147 (1985)], which is referred to as the CD18 family of glycoproteins. This family of glycoproteins consists of heterodimeric molecules that have an α-chain and a β-chain. Although the α chain is different for each antigen, the β chain is nearly identical [Sanchez-Madrid F. et al., J. Exper. Med. 158: 1785-1803 (1983). The β-chain of the glycoprotein family (CD18 family) has a molecular weight of 95 kd, while the α-chain has a molecular weight

HU 217 176 B

It varies between 150 and 180 kd [Springer, T .: Fed. Proc. 44: 2660-2663 (1985)]. Although the alpha subunit of membrane proteins does not exhibit such extensive homology as the β-subunits, a closer analysis of the α-subunits of glycoproteins revealed significant similarities. Similarities of the α and β subunits of glycoproteins related to LFA-1 are described by Sanchez Madrid, F. et al. Exper. Med. 158: 586-602 (1983); J. ExperMed. 158, 1785-1803 (1983)].

A group of patients are unable to express adequate members of the adhesion protein family on the surface of leukocyte cells [Anderson, D.C., et al., Fed. Proc. 44: 2671-2677 (1985); Anderson, D.C., et al., J. Infect. Dis. 152: 668-689 (1985)]. These patients suffer from leukocyte adherence deficiency disease (LAD) [Anderson, D.C., et al., Fed. Proc. 44: 2671-2677 (1985); Anderson, D.C., et al., J. Infect. Dis. 152: 668-689 (1985)]. LAD patients are characterized by soft tissue goiter lesions, limited plaque formation and wound healing, and abnormality of adhesion-dependent in vitro leukocyte function, and susceptibility to chronic and recurrent bacterial infections. Granulocytes from such LAD patients exhibit the same malfunctioning in vitro as normal counterparts in the presence of anti-CD18 monoclonal antibody. In particular, this means that they are unable to perform adhesion-related functions, such as attachment to endothelial cells. More importantly, the observation that these patients are incapable of a normal inflammatory response is due to the inability of the granulocytes to bind to the cellular substrate. Most important, however, is the observation that granulocytes from such LAD patients do not reach sites of inflammation, such as skin infection, because they are unable to attach to endothelial cells in the blood vessel near the inflammatory lesion. This interconnection is an important element in the formation of the sap.

Lymphocytes from such patients exhibit the same deficiencies in vitro as normal counterparts in which CD18 family molecules have been antagonized by antibodies. In addition, these patients are unable to elicit a normal immune response because the cells are unable to bind to the cellular substrate [Anderson, D.C., et al., Fed. Proc. 44: 2671-2677 (1985); Anderson, D.C., et al., J. Infect. Dis. 152: 668-689 (1985)]. These data suggest that immune responses are impaired when lymphocytes are unable to bind normally due to the lack of functional adhesion molecules of the CD18 family.

In conclusion, leukocytes can only maintain their health and viability if they are able to bind to other cells, such as endothelial cells. This binding requires cellular contact involving specific receptor molecules on the cell surface of leukocytes. These receptors ensure that the leukocyte binds to other leukocytes, endothelial cells or other non-vascular cells. Cell surface receptor molecules such as LFA-1, Mac-1 and p150.95 are closely related. Patients whose leukocytes do not contain these cell surface receptor molecules suffer from chronic and recurrent infections, as well as other clinical symptoms, such as "lack of antibody response."

In addition, since leukocyte binding is part of a process in which foreign tissue is recognized and excreted by the body, understanding the process can be particularly useful for organ transplants such as kidney transplants or non-solid organs such as bone marrow transplants, tissue transplants, and allergy and oncology.

HIV infection is the root cause of AIDS disease. Various variants of HIV are known, the two most important being HIV-1 and HIV-2. HTV-1 spread in North America and Europe, and HIV-2 spread only in Africa. Viruses have a similar structure and encode proteins with similar functions. It is believed that HIV infection occurs by attaching a viral protein (gp120 protein) to receptor molecules on the surface of T4 lymphocytes (T helper), such as CD4 molecules (Schnittmann SM et al., J. Immunol. 141: 4181-4). 4186 (1988)]. After binding to the receptor, the virus enters the cell, multiplies, and destroys the T cell. Therefore, the direct consequence of HIV infection is the death of the individual's T4 population.

Due to the destruction of T cells, the infected patient will be less able to overcome adaptive infections. Although cancer patients often develop cancers, the relationship between cancer and HIV infection is in most cases uncertain.

Although simple replication of the HIV virus is deadly to the infected cell, such replication is generally detectable only on a small fraction of the T4 cells of the infected patient. Recent studies suggest that viral infections are more common than previously assumed, and that cell infections can reach 1%.

Research has also revealed other mechanisms by which the HIV virus kills the T4 population.

In addition to HIV replication, HIV-infected cells may also be killed by cytotoxic killer cells. Killer cells are commonly found in the body and are responsible for the constant examination of the host for the destruction of foreign cells, such as those resulting from false blood transfusions or organ transplants. As a result of HIV infection, T4 cells bind to the gp120 molecule on the cell surface. Killer cells consider such a T4 cell as a foreign cell and, consequently, induce its destruction.

HIV infection can also cause the death of uninfected healthy cells. The infected cells deliver the gp120 protein into the bloodstream. Free gp120 molecules can bind to CD4 receptors on healthy, uninfected cells and thus appear to be HIV-infected. Cytotoxic killer cells recognize uninfected T4

EN 217 176 Β cell-bound gp120 molecule, the cell is considered foreign and triggers its killing.

Another mechanism of particular interest to the invention is that the HVV kills T4 cells during "syncytia" formation. These are multinucleated giant cells formed by fusion of several hundred T4 cells. As a result of HIV infection, the infected cell is more prone to fusion with other HIV-infected or healthy T4 cells. The resulting giant cell is dysfunctional and will soon die. Thus, in addition to HIV-infected cells, healthy T4 cells also die. This process is of particular interest to the present invention as it involves direct cell-to-cell contact of T4 cells. The formation of giant cells presupposes that such cells have the necessary conditions for fusion with healthy cells.

Thus, HIV infection, in particular HIV-1 infection, appears to affect cell surface expression of leukocyte integrin and heterodimeric integrin-mediated cellular binding reactions (Petit A.J. et al., J. Clin. Invest. 79, 188 (1987); Hidreth J. E. K. et al., Science, 244: 1075 (1989); A. Valentin et al., J. Immunology 144: 934-937 (1990); Ross D. R. et al., Trans Assoc. American Physicians 102: 117-130 (1989)]. After HIV-1 infection, homologous aggregation of U937 cells, i.e., cell surface expression of CDI8 and CDI1b, is enhanced [Petit A.J. et al., J. Clin. Invest. 79, 188 (1987)]. HIV-1 infected U937 cells bind more frequently to IL-1 stimulated endothelium than uninfected U937 cells, but this behavior can be suppressed when the infected cells are treated with anti-CD18 or anti-CDIIa monoclonal antibodies or the endothelial substrate is Treated with -ICAM-1 antibodies [Rossen RD et al., Trans. Assoc. American Physicians, 102: 117-130 (1989)]. Monoclonal antibodies to CD18 or CD11a also have the ability to inhibit the formation of syncytes involving phytohemagglutinin (PHA) stimulated lymphoblastoid cells and correspondingly infected CD4 negative T cells [Hildreth JEK et al., Science, 244, 1075 (1989)]. If only virus-infected cells are treated with anti-CD18 or anti-CDIIa monoclonal antibodies, syncytia formation is only slightly affected, suggesting that these antibodies primarily protect uninfected target cells from infection [Hildreth JEK et al., Science 244, 1075 (1989); Valentin, A., et al., J. Immunology 144: 934-937 (1990). In their cited paper, Valentin et al., Support this observation by demonstrating that CD18-specific monoclonal antibodies inhibit syncytia formation when co-cultured with continuous T-cell lines with HIV-1 infected U937 cells.

Although the mechanism by which CD18 or CDI lara-specific monoclonal antibodies protect sensitive cells from fusion with HIV-infected cells is not known at present, studies with the radiolabeled gp120 molecule suggest that heterodimeric molecules containing CD18 do not have a suitable binding site for the virus (Valentin, A., et al., J. Immunology, 144, 934-937 (1990)). Accordingly, HIV infection requires cell-cell interaction and / or virus-cell interaction that mimics such cell-cell interaction. One consequence of cell-to-cell interaction may be that cell-free virus or virus-infected cells pass through endothelial barriers. Virus-to-cell interactions, such as cell-cell interactions, may promote or allow free virus to bind to and / or infect healthy cells.

The present invention results in part from the observation that HIV infection, and in particular HIV-1 infection, enhances the expression of the CD11a / CD18 heterodimer and its binding ligand. This increased expression is important because it facilitates the binding or aggregation of HIV-infected T cells (i.e., homotypic aggregation). Since homotypic aggregation is not observed in resting normal leukocytes, this fact suggests that aggregation requires expression of CD11 / CD18 receptors and / or ligands such as ICAM-1. Homotypic aggregation requires LFA-1 to bind to ICAM-1. As mentioned above, ICAM-3 is the only representative of the ICAM family that is expressed at high levels on resting T cells. Without T-cell activation, only the antiICAM-3 antibody is able to inhibit T-cell binding to LFA-1. Therefore, the anti-ICAM-3 antibody can be used to inhibit T cell aggregation.

In addition, the anti-ICAM-3 antibody can be used to inhibit the adhesion process of infected T cells, which facilitates the transfer of HIV-1 from infected cells to healthy cells and promotes free virus infection of healthy cells.

Migration and dispersal of leukocytes play an important role in protecting the body from infection. However, the same processes are responsible for the migration and spread of virus-infected leukocytes. In particular, migration and dispersal of HIV-infected leukocytes play a special role. The migration of such cells results in the formation of extravascular foci, leading to tumors and other abnormalities.

Histological examination of the affected organs shows focal extravascular mononuclear cell infiltration. During the identification of virus-infected cells in such infiltrates in the central nervous system, HIV-1 infected cells were detected. Studies have shown that HIV-1 virus is primarily found in monocytes and macrophages and in related cells [R. T. Johnson et al., 1988, FASEB J. 2: 2970; Μ. H. Stoler et al., J. Amer. Med. Assn. 256: 2360 (1986); S. Gartner et al., Science 233: 215 (1986)].

The mechanism to stimulate extravascular infiltration of HIV-1 infected monocyte cells has not been previously known. Cell-free virus transport4 may be involved

Or transport of the virus through endothelial barriers in the cytoplasm of infected mononuclear cells.

Because HIV-1 infection promotes the binding of leukocytes to vascular endothelial cells and the cell surface expression of molecules that allow leukocytes to be translocated from the blood to extravascular tissue sites [C. W. Smith et al., J. Clin. Invest. 82, 1746 (1988), suggested the use of antibodies that inhibit the spread of HIV-infected cells by inhibiting cellular migration (International Patent Application Publication No. WO 90/13316).

Asthma is a heterogeneous group of different diseases. Typically, the trachea and bronchi are particularly sensitive to stimulation (Kay, A. B., Allergy and Inflammation, Academic Press, New York, USA, 1987). Clinically, asthma is characterized by extreme narrowing of the trachea and bronchi, accompanied by the appearance of dense sticky discharge, seizures with difficulty in breathing, coughing and wheezing. Although the relative contribution of each condition to the development of the disease is unknown, the result is an increase in airway resistance, an exaggeration of the lungs and chest, and an abnormal distribution of air exchange and pulmonary blood flow. During the course of the disease, acute symptoms appear occasionally, which are linked by asymptomatic periods. The acute stages result in hypoxia, which can be fatal. About 3% of the world's population suffers from asthma.

Two types of asthma are known, allergic asthma and idiosyncratic asthma. Allergic asthma is usually associated with inherited allergic diseases such as colds, hives and eczema. This condition is characterized by the intradermal injection of airborne antigens, such as pollen or environmental and occupational pollutants, into a positive bladder or reddening reaction and an increase in serum IgE. The development of allergic asthma in most patients is causally related to the presence of IgE antibodies. Asthma patients who do not have the above characteristics are defined as suffering from idiosyncratic asthma.

Allergic asthma is thought to be dependent on IgE regulated T and B lymphocytes and activates the interaction between airborne antigens and pre-formed IgE molecules bound to mast cells. In order to increase the patient's sensitivity, the antigen counterpart should be present at a concentration that provides IgE production for an appropriate period of time. After developing sensitivity, an asthmatic patient shows symptoms even at extremely low antigen concentrations.

Asthma symptoms can be aggravated by the presence of large amounts of triggering antigen, due to environmental factors, workplace factors, physical exertion and emotional stress.

Asthma can be treated with methylxanthine derivatives such as theophylline, β-adrenergic agonists such as catecholamine, resorcinol, saligenin and ephedrine, glucocorticoids such as hydrocortisone, agents that inhibit mast cell degranulation, such as chromones, atropine.

Asthma is believed to be associated with excessive penetration of eosinophils (eosinophilia) into lung tissue (Frigas, E., et al., J. Allergy Clin. Immunol. 77: 527-537 (1986)].

Immunologic basics of asthma by screening tests for focal pneumonia [Godard P. et al., J. Allergy Clin. Immunol. 70, 88 (1982)], and by examining respiratory smooth muscles deprived of epithelium (Flavahan, N.A., et al., J. Appl. Physiol. 58: 834 (1985); Bames, P.J., et al., Br. J. Pharmacol. 86, 685 (1985)]. Although these studies did not provide an explanation of the immune mechanism of asthma, they have established a generally accepted theory for the immunological behavior of the disease [Frigas, E., et al., J. Allergy Clin. Immunol. 77: 527-537 (1986)].

The most important feature of asthma pathology is massive eosinophil infiltration in the lung parenchyma and decreased mucociliary capacity. According to eosinophil theory, eosinophilic cells are transported to the bronchi to neutralize the harmful mediators released by the mast cells of the lungs. The theory is that eosinophils delivered to the bronchi are degranulated and release cytotoxic molecules. During degranulation, eosinophils release enzymes such as histaminase, arylsulfatase and phospholipase D, which enzymatically neutralize the malignant mediators of mast cells. However, these molecules promote the destruction of the mucociliary apparatus and thus prevent the emptying of the bronchial mucosa and contribute to the development of lung injury typical of asthma.

Because cell migration plays a role in asthma, it has been suggested to use antibodies that inhibit the migration of allergens (International Patent Application Publication No. WO 90/10453).

The present invention relates to a novel cellular adhesion molecule called intercellular adhesion molecule-3, abbreviated as ICAM-3. The present invention includes functional derivatives of ICAM-3, anti-ICAM-3 antibodies, fragments thereof, humanized anti-ICAM-3 antibodies, and other molecules capable of binding and inhibiting ICAM-3 function.

The present invention also relates to pharmaceutical compositions and diagnostic compositions containing the above molecules.

More particularly, the present invention relates to ICAM-3, substantially free of natural impurities, having a molecular weight of Mr = 124,000, determined by gel electrophoresis under reducing conditions, and Mr = 87,000, after treatment with N-glycanase, which binds to LFA-1.

The invention further relates to a recombinant DNA molecule encoding the above ICAM-3 or a functional derivative thereof.

HU217 176Β

The invention further relates to ICAM-3 or a functional derivative thereof for use as a medicament.

The invention further relates to a polyclonal or monoclonal antibody or antibody fragment capable of binding ICAM-3 or a functional derivative thereof for use as a medicament.

The invention further relates to a pharmaceutical composition comprising ICAM-3 or a functional derivative thereof, or a polyclonal or monoclonal antibody or antibody fragment or a derivative thereof which is capable of binding them, together with a pharmaceutical carrier or excipient.

Preferred is a pharmaceutical composition which can be used for therapeutic or prophylactic purposes to modify the biological function of a cell mediated by ICAM-3, preferably an inflammatory response, a hematopoietic tumor cell metastasis that requires a functional member of the CD-18 family to migrate with a virus, preferably HIV. modifying extravascular migration of virus-infected leukocytes or migration of cells associated with asthmatic response.

Further preferred is a pharmaceutical composition comprising ICAM-3 or a functional derivative thereof, or a polyclonal or monoclonal antibody or antibody fragment thereof or a toxin-derived derivative thereof, or a toxin-derived derivative of a CD18 family molecule or a functional derivative thereof. with the aim of suppressing infection of leukocytes with HIV, preferably HIV-1.

Further preferred is a pharmaceutical composition comprising ICAM-3 or a polyclonal or monoclonal antibody or antibody fragment thereof, or a toxin-derived derivative thereof, or a toxin-derived derivative of a CD18 family molecule or a functional derivative thereof, for use in therapy or prophylaxis. suppressing the growth of a tumor-expressing tumor cell.

Also preferred is a pharmaceutical composition comprising a toxin-derived derivative of ICAM-3 or a functional derivative thereof, which can be used for therapeutic or prophylactic purposes to suppress the growth of a tumor cell expressing LFA-1.

The present invention also provides a diagnostic composition capable of binding ICAM-3 or a functional derivative thereof, which comprises detectably labeled polyclonal or monoclonal antibody or antibody fragment.

The invention further provides a method for detecting a cell expressing ICAM-3 by:

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

b) detecting a nucleic acid molecule hybridized to a complementary nucleic acid molecule in the cell or cell extract.

The present invention further relates to a method for diagnosing inflammation in a mammalian patient or diagnosing the presence of ICAM-3 in a mammalian fluid sample by:

(a) incubating a sample of the patient with a composition comprising a polyclonal or monoclonal antibody or fragment thereof capable of binding ICAM-3 or a functional derivative thereof, and

b) detecting the fluid-bound antibody, wherein a sample of tissue is detected when inflammation is detected and blood, serum, plasma, joint fluid, amniotic fluid, spinal fluid, or urine are diagnosed when the presence of ICAM-3 in a fluid sample is diagnosed.

The invention further relates to a method for identifying an agent capable of modulating ICAM-3 binding to LFA-1 by contacting ICAM-3 with LFA-1 in the presence of the agent and measuring the ICAM-3 / LFA-1 binding. change.

The invention further relates to a process for the preparation of the above-mentioned ICAM-3 or a functional derivative thereof by isolating ICAM-3 from its natural source or by recombinant production and purification from its natural contaminants.

The present invention further relates to a method of producing a recombinant DNA molecule encoding the above ICAM-3 or a functional derivative thereof by recombinantly assembling a DNA molecule encoding ICAM-3 or a functional derivative thereof with another DNA molecule.

The invention further relates to a process for the production of a polyclonal or monoclonal antibody or antibody fragment capable of binding ICAM-3 and / or a functional derivative thereof by immunizing a host with ICAM-3 and / or an antigen-specific derivative thereof to ICAM-3 and / or. or antibodies capable of binding to its functional derivative are recovered from the host and optionally using polyclonal or monoclonal antibodies capable of binding to ICAM-3 and / or its functional derivative using cells producing the antibodies.

Figure 1 shows the binding of different cell lines to purified LFA-1. Binding of cell line (A) and lymphocytes (B) to purified LFA-1 is monitored by ICAM-1, ICAM-2 and ICAM-3. In doing so, BCECF-labeled cells are bound to LFA-1 coated microtiter plates in the presence of LFA-1 specific MAb blocker in the presence of ICAM-1, ICAM-2 and ICAM-3. A control without LFA-1 was used as a control.

Figure 2 is a flow cytometric analysis of cellular ICAM-1, ICAM-2 and ICAM-3 expression. In case (A), lymphoblastoid cell lines are saturated with MAb RR1 / 1 (anti-ICAM-1), MAb CB-IC2 / 1 (antiICAM-2), MAb CBR-IC3 / 1 (anti-ICAM-3) or non-binding. control as MAb X63 (thin line) followed by FITC anti-mouse immunoglobulin. In case (B), resting human leukocytes and 3-day PHA-activated lymphocytes are analyzed for ICAM expression as described above.

Figure 3 shows an immunoprecipitative assay of ICAM-3. In the case of (A), the lysate of ¹²⁵ I

As a non-binding control, we precipitated with X63 belonging to MAb CBIC3 / l (antiICAM-3). In case (B), a control that does not bind ¹²⁵ I-labeled SK.W3 cell lysate after (positive) or without (negative) treatment with N-glycanase MAb X63, MAb W6 / 32 (anti-HLA-A, B, C), MAb RR1 / 1 (anti-ICAM-1), MAb CBR-IC2 / 1 (antiICAM-2) or MAb CB-IC3 / 1 (anti-ICAM-3).

Figure 4 shows ICAM-3 immunoprecipitated from various sources. ICAM-3 was precipitated from ¹²⁵ I-labeled lymphocytes and neutrophil lysate using MAb-bound sepharose. The precipitated ICAM-3 is resolved by polyacrylamide gel electrophoresis. Other MAbs were used to precipitate other cell surface molecules: human Ig Sepharose (control), CBR-IC2 / 1 (ICAM-2), CBR-IC3 / 1 (ICAM-3), TS2 / 4 (LFA-1), LM2 / 1 (MAC-1).

Figure 5 shows the effect of PMA on the binding of SKW3 to ICAM-1 and ICAM-3. The assay investigates the ability of SKW3 cells to bind purified ICAM-1 and ICAM-3 and the stimulation of PMA for binding (Dustin et al., 1989, Nature 341: 619; Mariin et al., Cell 51: 813-819 (1987)]. In the figure, a representative assay is shown with standard deviation (SD).

Figure 6 shows antibody blocking of PMA-stimulated SKW3 cell binding. Various antibodies were tested to block the binding of PMA-stimulated SKW3 cells to purified ICAM-1 or ICAM-3 (Dustin et al., 1989, Natur. 341: 619; Mariin et al., Cell 51: 813-819 (1987)]. The figure shows the result of a representative assay and denotes SD.

Figure 7 shows binding of COS cell transfectants to purified ICAM. COS cells were transfected with the LFA-1 expression vector (deFougerolles et al., 1992, J. Exp. Med. 175: 185-190). Transfected cells are assayed for binding to immobilized ICAM-1 and ICAM-3 in the presence and absence of antiLFA-1 antibody (TS1 / 22). The figure shows the result of a representative assay and denotes SD.

Figure 8 shows the temperature dependence of PMA-stimulated SKW3 binding to ICAM. Binding of PMA-stimulated SKW3 cells to purified ICAM-1 and ICAM-3 was tested at 4, 16, 22 and 37 ° C (Mariin et al., Cell, 51, 813-819 (1987)). ]. The assay was performed in the presence and absence of LFA-1 MAb (TS1 / 22). The figure shows the result of a representative experiment and denotes SD.

Figure 9 shows the blocking of the PHA-stimulated T-cell division with antibody. Various antibodies block the division of PHA-stimulated T cells [Krensky et al., J. Immunol. 131: 611-616 (1983)].

Figure 10 shows the effect of anti-ICAM antibodies on a mixed lymphocyte reaction. Various antibodies inhibit the mixed lymphocyte reaction [Krensky et al., J. Immunol. 131,611-616 (1983)].

All. Fig. 3A shows a gas chromatogram of peptide fragments of ICAM-3. After purification, ICAM-3 is digested with Lys-C. The peptide fragments are resolved by high performance liquid chromatography.

Figure 12 shows clone 11.2. The positions of the NK-10 and NK-17 peptides, the various DNA sequences obtained, and the transmembrane region were indicated.

Figure 13 shows a comparison of the NK-10 primer-primed sequence with human ICAM-1 sequence. The assay identifies the homology between the NK-10 primer-initiated sequence and the human ICAM-1 protein by GAP program.

Figure 14 shows a comparison of the sequence initiated with the RM13 primer and the human ICAM -1 sequence. The assay identifies the homology between the sequence primed with the RM13 primer and human ICAM-1 by GAP program.

Figure 15 shows a comparison of the sequence initiated with the RM13 primer and the human ICAM-2 sequence. The assay identifies the homology between the RM13 primer sequence and the human ICAM-2 sequence by a GAP program.

Figure 16 shows a comparison of the sequence initiated with the T7 primer and the human ICAM-1 sequence. The assay identifies the homology between the T7 primer-initiated sequence and the human ICAM-1 sequence by GAP program.

Figure 17 shows a comparison of the sequence initiated with the T7 primer and the human ICAM-2 sequence. The assay identifies the homology between the sequence initiated by the T7 primer and the human ICAM-2 sequence using a GAP program.

Figure 18 shows a partial sequence of ICAM-3. The DNA sequence of clone 11.2 was determined by the usual method. Figure A shows the sequence obtained at the 5 'end of ICAM-3 obtained by treating clone 11.2 with T7 primer. Figure B shows the sequence obtained within the ICAM-3 gene obtained by treating clone 11.2 with the NK-10 sample. Figure C shows the 3'end sequence of ICAM-3 obtained by treatment of the reverse RM13 primer of clone 11.2.

The present invention is based on a previously unidentified binding ligand of LFA-1. Molecules involved in cellular adhesion, such as those belonging to the CD18 family, are referred to as adhesion molecules.

The LFA-1 leukocyte adhesion molecule mediates a broad spectrum of interactions between lymphocytes, monocytes, natural killer cells and granulocytes, and the immune system and other cells involved in the inflammatory process [Springer T. A. et al., Ann. Port. Immunol. 5: 223-252 (1987)].

LFA-1 is a receptor for ICAM-1, ICAM-2 and newly identified ICAM-3. These surface molecules are expressed on certain tissues and are induced by other tissues involved in inflammation (Mariin, S.D. et al., Cell, 51: 813-819 (1987); Dustin, M.L., et al., J. Immunol. 137, 245-254

HU 217 176 B (1986); Dustin, M.L., et al., Immunol. Today, 9, 213-215 (1988); and U.S. Patent Application Nos. 07/019440, filed February 26, 1987, and U.S. Patent Application Serial No. 07/250446, filed September 28, 1988).

LFA-1 is involved in both antigen-specific and antigen-independent T cytotoxic, T helper, natural killer, granulocyte, and monocyte interactions (Springer T. A. et al., Ann. Port. Immunol. 5: 223-252 (1987); Kishimoto T. K. et al., Adv. Immunol. (1988)].

ICAM-1 is a single-chain glycoprotein that varies in weight between different cell types and ranges from 76 to 114 kD. It is a member of the Ig superfamily and has five C-type domains [Dustin, M.L., et al., Immunol. Today, 9, 213-215 (1988); Staunton, D.E., et al., Cell 52: 925-933 (1988); Simmons, D., et al., Natura 331: 624-627 (1988). ICAM-1 is easily inducible by various cytokines such as IFN-g, TNF, and IL-1 on different cell types (Dustin, M.L., et al., Immunol Today, 9,213-215 (1988)). Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1-dependent adhesion of lymphocytes [Dustin, M.L., et al., J. Immunol. 137: 245-254 (1986); Dustin, M.L., et al., J. Cell. Biol. 107: 321-331 (1988); Dustin, M.L., et al., J. Exp. Med. 167: 1323-1340 (1988). Adhesion is blocked when lymphocytes are pretreated with LFA-1 MAb or other cells are treated with ICAM-1 MAb [Dustin, M.L., et al., J. Immunol. 137: 245-254 (1986); Dustin, M.L., et al., J. Cell Bioi. 107: 321-331 (1988); Dustin, M.L., et al., J. Exp. Med. 167: 1323-1340 (1988). Similar results obtained with purified ICAM-1 on an artificial membrane or in a Petri dish confirm that LFA-1 and ICAM-1 are receptors to each other [Mariin, S.D. et al., Cell. 51: 813-819 (1987); Makgoba, M.W., et al., Natura 331: 86-88 (1988). For the sake of clarity, these are referred to hereinafter as the receptor and the ligand. Further details of ICAM-1 are found in U.S. Patent Nos. 07/045,963, 07/115798, 07/155943, 07/189815, and 07/250446.

Other LFA-1 ligands were also believed to exist alongside ICAM-1 [Rothlein R. et al., J. Immunol. 137: 1270-1274 (1986); Makgoba, M.W., et al., Eur. J. Immunol. 18: 637-640 (1988); Dustin, M.L., et al., J. Cell. Biol. 107,321-331 (1988)]. The second identified LFA-1 ligand is designated ICAM-2.

ICAM-2 differs from ICAM-1 in cellular distribution and in the fact that it is not inducible by cytokine. ICAM-2 has an integral membrane protein with two Ig-like domains, whereas ICAM-1 has five Ig-like domains (Staunton, D.E. et al., Cell 52: 925-933 (1988); Simmons, D., et al., Natura 331: 624-627 (1988). Interestingly, ICAM-2 is much more similar to the two N-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 to other members of the Ig superfamily, which is Ig-like. refers to a subset of ligands that bind to the same receptor family. ICAM-2 is described in more detail in U.S. Patent Application Serial No. 07 / 454,294.

We have found that there is a third LFA-1 ligand designated ICAM-3.

During the development of MAb against ICAM-2, several previously LFA-1-dependent and ICAM-1-independent phenomena can be analyzed, suggesting the existence of a third LFA-1 ligand. Binding of purified LFA-1 to various cell types such as epithelial and endothelial cells can be completely inhibited by the combination of ICAM-1 and ICAM-2 MAb, whereas in several lymphoid cell lines, such as the SKW3 T cell lymphoma cell line, there is an LFA-1 adhesion pathway. , which is ICAM-1 and ICAM-2 independent (Figure 1).

To characterize the above version of the adhesion process, an MAb for SKW3 is generated and, similarly to anti-ICAM-1 and anti-ICAM-2 MAb, the extent to which the cell line binds to purified LFA-1 is tested. A MAb, CBR-IC3 / 1, was selected which completely inhibits this adhesion variant (Figure 1). The binding of SKW3 cells to purified LFA-1 is only slightly inhibited by the combination of blocking anti-ICAM-1 and anti-ICAM-2 MAbs. Even when used alone, the antiICAM-3 MAb, CB-IC3 / l, significantly inhibits adhesion, and this inhibition can be completed in combination with the blocking anti-ICAM-2 MAb. This means that the adhesion of SKW3 to purified LFA-1 is largely mediated by ICAM-3 and partially by ICAM-2. During binding to LFA-1, each of the four cell lines tested uses different levels of the three ICAM molecules, suggesting different processes of MAb inhibition. Adhesion of the JY B lymphoblastoid cell line is primarily via the ICAM-1 pathway and is only marginally affected by the ICAM-2 and ICAM-3 pathways. Another B-lymphoblastoid cell line, the SLA cell line, utilizes both the ICAM-1 and ICAM-3 pathways, since adhesion alone is not inhibited by either ICAM-1 or ICAM-3 MAb but completely blocked by the two MAb together. The thymus tumor cell line, Jurkat, uses the ICAM-2 and ICAM-3 pathways during adhesion and is only slightly affected by the ICAM-1 pathway. Resting T-lymphocytes and their phytohemagglutinin (PHA) -activated form were also examined (Fig. 1B). Resting lymphocytes bind strongly to purified LFA-1 (Dustin et al., Natur. 341, 619-624 (1989)) and this binding is predominantly ICAM-3 dependent. Upon PHA activation, expression of ICAM-1 is induced and binding to LFA-1 occurs primarily via the ICAM-1 pathway and is only partially mediated by the ICAM-3 pathway. The ICAM-2 component is detectable by binding to both dormant and activated T cells, but the presence of ICAM-1 and ICAM-3 obscures it. ICAM molecules are used in excess because each cell tested has at least two ICAMs

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MAb is required to achieve adequate inhibition. The only exception is the adhesion of resting lymphocytes to LFA-1, which is predominantly ICAM-3 dependent. In all cases, the combination of the three anti-ICAM MAb inhibits the binding of LFA-1 to a level comparable to that obtained with the presence of the anti-LFA-1 MAb.

The relative activity of the three ICAMs relative to LFA-1 can be assayed by comparing their involvement in LFA-1 binding, which can be accomplished by immunofluorescence flowing cytometric measurement of cell surface expression (Figure 2). Comparison of the surface expression of ICAM molecules and their involvement in LFA-1 binding shows that ICAM-1 shows the highest affinity for LFA-1, whereas ICAM-2 and ICAM-3 have similar but lower affinities. For example, Jurkat cells express ICAM-2 and ICAM-3 at similar levels and are both involved in binding LFA-1. Where the expression of ICAM-2 is higher than that of ICAM-3, so in JY cells, the ICAM-2 pathway in LFA-1 binding is higher than that of ICAM-3. In contrast, SLA and SKW3 cells express three to five times more ICAM-3 than ICAM-2, and thus, not surprisingly, the ICAM-3 pathway dominates adhesion at the expense of the ICAM-2 pathway. In resting T-lymphocytes, where ICAM-3 is well expressed and ICAM-1 is absent, binding of LFA-1 is mainly mediated by ICAM-3. If ICAM-1 is expressed as in SLA-1, JY and PHA-activated T cells, it is the primary pathway.

The distribution pattern of ICAM-3 differs from ICAM-1 and ICAM-2 at several points. Unlike ICAM-1 and ICAM-2, ICAM-3 is not expressed on resting or stimulated endothelium, consistent with the observation that LFA-1-dependent binding of cells to resting and stimulated endothelium is dependent on ICAM. -l and ICAM-2. ICAM-3 is confined to the hematopoietic lineage because, with some exceptions, it is highly expressed on lymphoid and monocyte cell lines. In all cases, however, expression of ICAM-3 on the cell line can be coordinated with the LFA-1-dependent and ICAM-1, ICAM-2-independent pathways of adhesion. Cell lines that bind LFA-1 only through ICAM-1 and ICAM-2-η, such as HUVEC and Raji, do not express ICAM-3, whereas these cell lines (JY, U937 and Sup T) , which show poor binding to this third pathway, exhibit correspondingly low ICAM-3 surface expression.

ICAM-3 is significantly different from ICAM-1 and ICAM-2 in terms of leukocyte expression (Figure 2B). ICAM-3 is expressed in large amounts on resting lymphocytes, monocytes, and neutrophils, while expression of ICAM-1 and ICAM-2 is negligible or absent. In comparison, ICAM-1 and ICAM-2 are poorly expressed on monocytes and only ICAM-2 is found on resting lymphocytes. Neither ICAM-1 nor ICAM-2 is expressed on neutrophils. Upon activation of lymphocytes with PHA, the expression of ICAM-3 is increased 2 to 3-fold, whereas the expression of ICAM-1 is significantly induced [Dustin et al., J. Immunol. 137, 245-254 (1986)] (Figure 2B).

Immunoprecipitated assays of ICAM-3 from various ¹²⁵ I-labeled cell lines show a sharp band under reducing conditions at a molecular weight of 124,000 and only slightly increase mobility under non-reducing conditions (Fig. 3A). By treatment with N-glycanase, the band of ICAM-3 is reduced to a molecular weight of 87,000, indicating that ICAM-3, like ICAM-1 and ICAM-2, is a highly glycosylated protein (Fig. 3B). Biochemical characteristics, forms of expression and functional properties distinguish ICAM-3 from previously known adhesion molecules, such as the human self-directed LAM-1 receptor [Tedder et al., J. Immunol. 144: 532-540 (1990)], an inducible endothelial adhesion molecule, VACAM-1 (Rice et al., J. Exp. Med. 171, 1369-1374 (1990)); Carlos et al., Blood (1990); Osbom et al., Cell, 59, 1203-1211 (1989)] and from the VLA family of matrix receptors [Hemler Μ. E .: Ann. Port. Immunol. 8, 365-400 (1990)] and no MAb with similar cellular distribution was found in the four leukocyte work databases (Gilks, WR et al., Leukocyte Typing Data Base IV, Oxford University Press, Oxford, England, 1990).

The existence of the three LFA-1 ligands suggests that LFA-1-dependent leukocyte interactions are specialized in various aspects. ICAM-1 is predominantly expressed on the endothelium and other epithelial cell types and is strongly induced by inflammation and immune responses where it regulates cellular localization and promotes specific antigen recognition [Wawryk et al., Immunol. Port. 108: 135-161 (1989)]. Because ICAM-2 is the dominant LFA-1 ligand in resting endothelium, this pathway of adhesion may play an important role in the normal circulation of LFA-1-bearing lymphocytes through the tissue endothelium [Hamann et al., J. Immunol. 140: 693-699 (1988); Mackay et al., J. Exp. Med. 171, 810-817 (1990); Pals et al., J. Immunol. 140: 1851-1853 (1988); Nunoi et al. Path. 19: 753-759 (1988)]. The function of ICAM-3 on neutrophils is currently unknown, since homotypic aggregation of neutrophils is primarily Mac-1 dependent, despite the presence of LFA-1 (Anderson et al., J. Immunol. 137: 15-27 (1986); Patarroyo et al., Scand. J. Immunol. 22: 619-631 (1985). The finding that dormant T-lymphocytes bind to LFA-1 is primarily through ICAM-3-οη, combined with the fact that ICAM-3 is much more expressed on monocytes and dormant lymphocytes than other LFA-1s. ligand, these molecules play an important role in triggering the immune response. In line with this, adhesion of T cells to antigen producing cells requires an LFA-1: ICAM interaction [Dransfield et al., Imm. Port. 114: 29-44 (1990); Makgoba et al., Immunol. Today 10, 417-422 (1989)] and thus ICAM-3 can play an important role there,

EN 217 176 Β, where neither ICAM-1 nor ICAM-2 are expressed properly. Consistent with this is the observation that LFA-1 ligands other than ICAM-1 play an important role in allogeneic and autologous mixed lymphocyte reactions [Bagnasco et al., Cell Immunol. 128: 362-369 (1990)]. In addition, ICAM-3 is believed to play an important role in antigen-specific interactions between T and B lymphocytes when only one of these lymphocyte types is in activated form. ICAM-3 may also be important in the destruction of T cell targets that are LFA-1 dependent but are ICAM-1 independent [Makgoba et al., Eur. J. Immunol. 18.637 to 640 (1988)].

The presence of multiple ICAMs facilitates clinical treatment with the MAb for ICAM and its analogs. ICAM-1 MAb can be effectively used in realistic [Cosimi et al., J. Immunol. 144,4604-4612 (1990)] and cardiac [Flavin et al., Transplant Proc. 23, 533-534 (1991) for the in vivo delay of allografts. ICAM-3 MAb inhibits different and overlapping immune responses under in vivo conditions by inhibiting LFA-1-dependent adhesion interactions of different cell types.

The cloning of the ICAM-3 gene can be accomplished by any conventional method. An alternating vector library of cDNA inserts derived from cells expressing ICAM-3at can be analyzed for the presence of an insert containing the ICAM-3 gene. Such an assay can be accomplished by transfecting cells with a vector and assaying for expression of ICAM-3.

The ICAM-3 cDNA is readily identified by the modified method of Aruffo and Seed (Seed, B. et al., Proc. Natl. Acad. Sci. USA 84: 3365-3369 (1987), which identifies ligands for adhesion molecules. In this method, a cDNA library is prepared from cells expressing ICAM-3, such as the SLA, Jurkat or SKW3 lymphoblastoid cell line. The cDNA library is preferably prepared from T cells. This library is used to transfect cells that generally do not express ICAM-3, such as the Cos or HeLa cell line. Transfected cells are plated in Petri dishes previously coated with LFA-1 or anti-ICAM-3 antibodies. Transfected cells containing the ICAM-3 coding sequence and expressing this ligand on the cell surface bind to LFA-1 or anti-ICAM-3 antibodies on the surface of the Petri dish. Unbound cells were washed and bound cells were removed from the Petri dish and cultured. Recombinant ICAM-3 expressed by the cells is isolated and sequenced.

When LFA-1 is used in the Petri dish, anti-ICAM-1 and anti-ICAM-2 specific antibodies are added to the Petri dish to inhibit the binding of cells capable of expressing ICAM-1 or ICAM-2. The binding of ICAM-1 + or ICAM-2 + transfectants to the LFA-1 coated plastic is inhibited by antibodies such as RR1 / 1, anti-ICAM-1 MAb and CBR-IC2 / 2, anti-ICAM-2 MAb. Binding of ICAM-3 transfected cells to LFA-1 coated Petri dishes is inhibited by EDTA and antiLFA-1 MAb without the use of anti-ICAM-1 or anti-ICAM-2 MAb. Therefore, ICAM-1 or ICAM-2-expressing cells are unable to bind to the Petri dish and are washed away with the non-bound cells. In this way, cells capable of expressing ICAM-3 are enriched.

Alternative methods for obtaining gene sequences capable of encoding ICAM-3 are known in the art and readily utilized by those skilled in the art.

The construction of gene sequences suitable for coding for ICAM-3 can be accomplished with an oligonucleotide sample that is used to screen a cDNA or genomic DNA library. Preferably, the ICAM-3 protein is purified by immuno-purification procedures and the terminal amino acid sequence is determined in the usual manner. Alternatively, the ICAM-3 protein may be enzymatically cleaved using, for example, trypsin or Lys-C, the peptide purified, and the amino acid sequence of the internal fragments determined.

After determining the partial amino acid sequence, an oligonucleotide sample is prepared based on the codons preferred by the organism used, a degenerate sample is prepared with all possible codon combinations, or codons homologous to known ICAM-1 or ICAM-2 DNA sequences, and . The probe obtained is used to examine genomic or cDNA libraries for the presence of probe hybridizing sequences.

Methods identical or similar to those described above can be effectively applied to human aldehyde dehydrogenase [Hsu L. C. et al., Proc. Natl. Acad. Sci. USA 82, 3771-3775 (1985)], fibronectin [Suzuki S. et al., Eur. Mol. Biol. Organ. J. 4: 2519-2524 (1985)], the human estrogen receptor gene (Walter P. et al., Proc. Natl. Acad. Sci. USA 82,7889-7893 (1985), and tissue-type plasminogen activator (Pennica D. et al., Natur. 301, 214-221 (1983)).

Another alternative to cloning the ICAM-3 gene is to generate an expression library from cells capable of expressing ICAM-3 by cloning genomic DNA or preferably cDNA. The library is then assayed for expression of anti-ICAM-3 binding protein.

The cloned ICAM-3 gene obtained by one of the methods described above is operably linked to an expression vector and introduced into a bacterial or eukaryotic cell to produce the ICAM-3 protein. These steps are readily accomplished by the well-known manual of Manniatis T. et al., And by standard procedures.

Cloning of the ICAM-3 gene can also be accomplished by PCR assuming that ICAM-3 is homologous to ICAM-1 and ICAM-2. The degenerate oligonucleotide primer based on the common sequences of ICAM-1 and ICAM-2 can be used from cells capable of expressing the ICAM-3 protein.

HU 217 176 B

CDNA or mRNA polymerase from SKW3 almond cell line (deFougerolles et al., J. Exp. Med. 175: 185-190 (1992)). The resulting clones were sequenced and sequences other than ICAM-1 and ICAM-2 were used to generate full-length cDNA clones.

In the methods described above, clone validation can be accomplished by expressing a full-length clone, for example, in COS cells and assaying with an ICAM-3 MAb.

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

The fragment of ICAM-3 can be any polypeptide that is a subset of the parent molecule. Preferred are soluble ICAM-3? Fragments having original activity of ICAM-3 and soluble (i.e., non-membrane bound). Preferably, the soluble moiety is prepared from the parent molecule by bypassing the membrane bridge or by deleting the hydrophilic amino acid bristles or replacing them with hydrophobic moieties. The selection of appropriate groups is within the skill of the art.

Fragments comprising any portion of the complete Ig-like domain, such as the D1, D1 and D2, D1-3, D1-4 and D1-5 domains are also preferred.

ICAM-3 is a molecule that is substantially similar in structure and function to the parent molecule or fragment thereof.

One molecule is considered to be "substantially similar to another molecule if both molecules exhibit a high degree of structural similarity or both molecules exhibit similar biological activity. Accordingly, when two molecules exhibit similar activity, they are referred to as variants in the terms used herein, even if the structure of one molecule is not found in the other molecule or the amino acid sequences are not identical.

As used herein, a molecule is considered to be a chemical derivative of another molecule if it contains additional chemical moieties that do not generally occur in the molecule of natural origin. These details can improve the solubility, absorption, biological half-life and other similar properties of the molecule. These details may reduce the toxicity of the molecule or eliminate or attenuate unwanted side effects. Details with such properties can be found in Remington's Pharmaceutical Sciences (1980).

A toxin derivative of a molecule is a special class of chemical derivatives. A toxin derivative is a molecule, such as ICAM-3 or anti-ICAM-3 antibody, which is covalently attached to a toxin moiety. The binding of such a moiety to the molecule takes place in a conventional manner.

When a molecule binds to a cell, the toxin moiety is brought close to the cell and promotes its destruction. Any known toxin may be used as the toxin moiety, but ricin toxin, cholera toxin, diphtheria toxin, radioisotope toxins and membrane channel toxins are preferred.

Functional derivatives of ICAM-3 containing up to 100 residues can be readily prepared by in vitro synthesis. If desired, such fag fragments may be modified in a conventional manner by reacting selected amino acid residues of the purified or crude protein with an organic derivatizing agent which is reactive with the particular side chain or terminal residue. The resulting covalent derivative can be used to identify residues that are important for biological activity.

Fragments of ICAM-3 can be prepared and isolated by various methods. During derivatization with bifunctional agents, ICAM-3 may be bound to a water insoluble carrier matrix. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates or the reactive substrates disclosed in U.S. Patent Nos. 3,969,287, 3,691,106, 4,195,128, 4,227,537, and 4,330,440 may be used to immobilize the protein. .

The functional derivative of ICAM-3 has an altered amino acid sequence, which can be produced, for example, by mutating the DNA encoding ICAM-3. Such a functional derivative can be formed, for example, by deleting residues from the amino acid sequence of ICAM-3, introducing residues, or substituting residues. The final structure can be combined in any way with the deletion, insertion, and substitution, provided that the final structure has the desired activity. It will be appreciated that mutations in the DNA encoding the functional derivative are not located outside the reading frame and preferably do not form complementary regions leading to the formation of a secondary mRNA structure (EP 75445).

At the genetic level, functional derivatives can be produced by site-specific mutation of the DNA encoding ICAM-3, resulting in DNA encoding the functional derivative, which can be expressed in recombinant cell culture.

While the amino acid sequence alteration site is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of a site-directed mutation, a random mutation at a targeted codon or stretch, such as a linker-directed mutation, can be used, generating a large number of derivatives that can be expressed and tested for an optimal combination of desired activity. Mutation at predetermined positions of a DNA can be accomplished by conventional means, such as site-specific mutation.

Accordingly, functional derivatives of ICAM-3 may advantageously be produced by site-specific mutation of DNA encoding ICAM-3 or a previously produced functional derivative of ICAM-3. Place11

Specific mutation B is performed as usual (Maniatis T. et al., Molecular Cloning, A Laboratory Manual, Coldspring Harbor, New York, USA, 1982). By site-specific mutation, functional derivatives of ICAM-3 can be obtained via specific oligonucleotide sequences encoding the DNA sequence corresponding to the desired mutation.

A site-specific mutation can be used to delete, insert or replace amino acids. The deletion in the amino acid sequence generally encompasses about 1-30 residues, preferably 1-10 residues, and generally encompasses adjacent residues. Preferably, a bypass is obtained that produces a soluble form of ICAM-3. Preferably, soluble forms of ICAM-3 can be produced by bypassing the membrane bridge portion of ICAM-3 or by eliminating hydrophobic residues within ICAM-3-οη.

During amino acid insertion, one or more amino acids are inserted into the coding sequence of ICAM-3 or the polypeptide of substantially infinite length is inserted by terminal fusion. Intraspecific insertion (i.e., insertion within the entire ICAM-3 molecule sequence) generally extends to about 1-10 residues, preferably 1-5 residues. An example of a terminal insertion is a fusion with a signal sequence, which may be heterologous or homologous to the host and which promotes secretion from the recombinant host when linked to the N-terminal portion of the molecule.

A third group of functional derivatives are those in which one or more amino acid residues within the ICAM-3 molecule are removed and replaced with a different residue. Such substitution is preferably carried out according to the following table, whereby ICAM-3 having modified properties is obtained.

Table 1

Original residue Substitute residue below gly, ser Arg lys Asn gin, his asp glu Cys ser Gin asn Glu asp Gly area, pro His asn, gin Ile leu, hours Leu ile, hours Lys arg, gin, glu Met leu, tyr, ile Phe met, leu, tyr Ser thr

Original residue Substitute residue Thr ser Trp tyr Tyr trp, phe With ile, leu

Functional or immunological identity may be substantially altered by selecting substitutions that are less conservative than the residues in Table 1, i.e. residues that differ more in their effect.

a) preserving the structure of the polypeptide chain in the substitution environment, for example in a flat or helical conformation,

(b) preserving the charge or hydrophobic nature of the molecule; or

(c) preserving side chains.

Examples of less conservative substitutions include

(a) glycine and / or proline have already been replaced or bypassed or inserted by amino acids,

b) replacing the hydrophilic residue with a hydrophobic residue,

c) replacing the cysteine residue with another residue,

d) replacing an electropositive residue with an electronegative charged residue, or

e) replacing the residue having a large bulky side chain with a residue without side chain.

Substitution which affects the solubility of ICAM-3 is preferred. This is most easily accomplished by replacing hydrophilic amino acids with hydrophobic amino acids.

For example, a mutation to increase the affinity of ICAM-3 may be accomplished by introducing residues at the homologous positions of ICAM-1 or ICAM-2. Similarly, mutant ICAM-3 molecules which lack the CHO moiety attached to the nitrogen atom at positions homologous to ICAM-1 or ICAM-2 can be prepared.

It is difficult to determine exactly how a particular substitution, deletion, or insertion affects the biological activity of ICAM-3. However, one skilled in the art will recognize that this can be determined by routine comprehensive testing. For example, the derivative is generally prepared by a specific mutation of a site directed by a DNA linker encoding the native ICAM-3 molecule. The derivative is then expressed in a recombinant host and optionally purified from the cell culture, for example, by immunoaffinity chromatography.

The effect of the cell lysate or purified derivative is then tested by assay for the desired property. For example, the change in the immunological character of a functional derivative, such as its affinity for a particular antibody, can be measured by competitive type immunoassay. The change in the immunomodulatory effect can also be measured by appropriate testing.

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Changes in protein properties such as redox, thermal stability, biological half-life, hydrophobicity, susceptibility to proteolytic degradation, or binding to a carrier or aggregation to a multimeric molecule can be assayed in the conventional manner.

An ICAM-3 agonist is a compound that provides or enhances the ability of ICAM-3 to induce biological functions. An example of such an agonist is a substance which promotes the binding of ICAM-3 to the cellular receptor.

An antagonist of ICAM-3 is a compound which abolishes or reduces the ability of ICAM-3 to perform a biological function. Examples of such agonists include ICAM-1 and ICAM-2, functional derivatives thereof, and anti-ICAM-3 antibody and anti-LFA-1 antibody.

The cellular aggregation assay disclosed in U.S. Patent Nos. 07/045963, 07/115798, 07/155 943, 07/189815 or 07/250446 can be used to measure LFA-1-dependent aggregation and thus identify substances that affect ICAM. -3 / LFA-1 aggregation. That is, this assay can be used to identify ICAM-3 agonists and antagonists. Antagonists may decrease the ability of LFA-1 or ICAM-3 to mediate aggregation. In addition, non-immunoglobulin (i.e., chemical) substances can be assayed for the above assay to determine whether they agonize or antagonize ICAM-3 / LFA-1 mediated aggregation. Such aggregation assays are generally performed on PMA-stimulated peripheral T cells. This implies that the binding of peripheral blood cells to LFA-1 can be used to assay for ICAM-3 mediated aggregation.

As an immunoglobulin antagonist, the antibody specific for ICAM-3, such as CBR-IC3 / 1, is preferably used in the present invention. Other appropriate anti-ICAM-3 antibodies may be prepared by various methods.

The antibody specific for ICAM-3 or functional derivatives thereof may be polyclonal or monoclonal. In addition, the antibody of the invention can be humanized in the usual manner (International Patent Applications PCT / US91 / 02942 and PCT / US91 / 02946).

For example, the anti-ICAM-3 antibody can be prepared by introducing ICAM-3 or a peptide fragment thereof into a suitable animal. The immunized animal produces a polyclonal antibody upon treatment. Using the peptide fragment of ICAM-3, a specific antibody sequence is generated which reacts with epitopes in the peptide fragments used to immunize the animal.

Alternatively, anti-ICAM-3 antibodies can be generated using naturally occurring lymphocyte surface expression of ICAM-3. Such cells are introduced into a suitable animal, for example by intraperitoneal injection, which produces antibodies capable of binding a molecule of the ICAM-3 or CDI8 family. If desired, the serum of such an animal may be removed and used to produce polyclonal antibodies capable of binding the molecules.

Alternatively, anti-ICAM-3 antibodies can be produced by adapting the Selden method (European Patent Publication No. 289034 and Selden RF et al., Science, 236: 714-718 (1987)). 3 molecules or fragments thereof are transfected with an expression vector which transmits an ICAM-3, which elicits an immune response in the animal, leading to the production of anti-ICAM-3 antibodies.

Preferably, however, splenocytes are removed from any animal immunized to obtain a monoclonal antibody capable of binding ICAM-3.

The hybridoma cells obtained as described above can be assayed by various methods to identify hybridoma cells that select for antibody binding to ICAM-3. The assay preferably identifies the molecule by its ability to inhibit aggregation of cells capable of expressing ICAM-3 and unable to express ICAM-1 and ICAM-2. Antibodies to aggregation are further tested to determine that aggregation is inhibited by binding to ICAM-3 or molecules of the CD18 family. Any device capable of discriminating between ICAM-3 and CDI8 family molecules can be used. For example, the antibody-bound antigen can be analyzed by immunoprecipitation or by polyacrylamide gel electrophoresis. Antibodies that bind molecules of the CDI8 family can be distinguished from antibodies that bind ICAM-3 by measuring the binding of the antibody to cells capable of expressing LFA-1 but unable to express ICAM-3 (or vice versa). Binding of an antibody to a cell capable of expressing LFA-1 but unable to express ICAM-3 can be detected by conventional means. Examples include immunoassay (e.g., immunofluorescence assay), cellular agglutination, filter binding assay, and antibody precipitation assay.

Preferably, the antibody is selected for binding to cells capable of expressing ICAM-3 but not for cells unable to express ICAM-3.

In addition to the above-mentioned agonists and antagonists of ICAM-3, the present invention may be used to treat inflammation, HIV infection and asthma, for example, anti-ICAM-3 antibodies and receptor molecules capable of binding ICAM-3, and fragments thereof.

These anti-idiotypic antibodies bind (or block) to ICAM-3 in a competitive manner. Such antibodies may be prepared, for example, by preparing an antibody specific for an antiICAM-3 antibody and assaying its binding to the natural binding ligands of ICAM-3.

Because members of the CDI8 family are capable of binding ICAM-3, the use of such molecules (e.g.

Β a heterodimer with α and β subunits, or in the form of a molecule consisting only of or only β subunits, or fragments thereof), initiates competition between molecules and cells for binding to ICAM-3 on the cell.

The anti-aggregation antibodies of the invention can be identified and titrated in any manner. For example, the differentiation of antibodies in binding to cells expressing ICAM-3, such as T cells, and the extent to which they are unable to bind cells that do not express ICAM-3, can be examined. For example, the methods described in U.S. Patent Application Nos. 07/045963, 07/115 798.07 / 155 943, 07/189815 or 07/250446 may be used to measure cellular aggregation. The binding of antibodies to ICAM-3 or a peptide fragment thereof can also be measured. It will be apparent to those skilled in the art that the above assays may be modified or combined in any manner to assay different properties and to discriminate ICAM-3 binding antibodies from molecules of the CD18 family.

The agents of the invention may be prepared by natural methods such as inducing the production of a non-immunoglobulin antagonist of ICAM-3 in an animal, plant, fungus or bacterium, or by inducing the production of polyclonal antibodies to bind ICAM-3 in an animal; and synthetic methods such as synthesizing ICAM-3, a functional derivative thereof, or a protein antagonist such as an immunoglobulin or a non-immunoglobulin antagonist; hybridoma technology, such as the production of a monoclonal antibody capable of binding ICAM-3; or by recombinant technology, for example, by producing a recombinant plasmid or viral vector in a variety of host organisms such as yeast, bacteria, fungi or cultured mammalian cells. The method is selected depending on various factors, such as suitability or yield. It is also possible to combine two or more of the above technologies to produce the desired anti-inflammatory agent.

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

One embodiment of the present invention is based on the interaction of ICAM-3 and its functional derivatives with molecules of the CD18 family, in particular LFA-1. The interaction between ICAM-3 and glycoproteins of the CD18 family can be used to suppress, i.e., prevent and attenuate inflammation.

By inflammation is meant the reactions of the specific defense system and the non-specific defense system.

A specific defense system is a component of the immune system that responds to the presence of specific antigens. Inflammation is considered to be the response of a specific defense system when inflammation is caused, mediated, or associated with the reaction of a specific defense system. Examples of inflammation from the response of the specific defense system are antigens, such as rubella virus response, autoimmune diseases, and T-cell-mediated, delayed-type hypersensitivity response (e.g., patients with a positive result in the Mantaux test). Other examples of inflammation resulting from reactions of the specific defense system include chronic inflammatory disease and rejection following transplantation of solid tissues and organs such as kidney and bone marrow.

The reaction of the non-specific defense system is the reaction mediated by leukocytes without immunological memory. Such cells include granulocytes and macrophages. Inflammation is considered to be a response to a non-specific defense system if it is caused, mediated, or associated with a non-specific defense system. Examples of inflammation resulting at least in part from the reaction of a non-specific defense system include inflammation associated with conditions such as adult respiratory distress syndrome (AR.DS), blood poisoning, multiple organ injury caused by trauma or bleeding, myocardial or other tissue reperfusion injury, reactive arthritis, dermatosis with an acute inflammatory component, acute purulent meningitis, or other inflammatory lesions of the central nervous system such as stroke, heat injury, hemodialysis, leucapheresis, ulcerative colitis, ulcerative colitis, ulcerative colitis, ulcerative colitis, cytocin toxicity.

As mentioned above, binding of ICAM-3 to CD18 family molecules plays a central role in cellular adhesion. Through the process of adhesion, lymphocytes constantly monitor the presence of foreign antigens in the body. Although these processes are generally beneficial and therefore desirable, they may result in rejection after solid organ transplantation, such as kidney transplantation, and non-solid organ transplantation, such as bone marrow or tissue transplantation, and are a trigger for several autoimmune diseases. Therefore, any device suitable for attenuating or inhibiting cellular adhesion may be advantageous after a solid organ, such as a kidney transplant, as well as a non-solid organ, such as a bone marrow transplant, and in autoimmune patients.

Monoclonal antibodies specific for CDI8 molecules inhibit various adhesion-dependent functions of leukocytes, such as endothelial binding (Haskard, D., et al., J. Immunol. 137: 2901-2906 (1986), homotypic adhesion (Rothlein R. et al., J. Exp. Med. 163, 1132-1149 (1986)), antigen and mitogen-induced proliferation of lymphocytes (Davignon, D. et al., 1986). Proc. Natl. Acad. Sci. USA 78: 4535-4539 (1981)], antibody formation [Fischer A. et al., J. Immunol. 136: 3198-3203 (1986)] and effector functions of leukocytes, such as the destructive effect of cytotoxic T cells [Krensky A.M. et al., J. Immunol.

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132, 2180-2182 (1984)], the destructive effect of macrophages [Strassman, G., et al., J. Immunol. 136: 4328-4333 (1986)] and the destructive action of all cells involved in antibody-dependent cellular cytotoxic reaction (Kohl, S. et al., J. Immunol. 133: 2972-2978 (1984)]. In the above functions, the antibody inhibits the binding of leukocytes to the appropriate cellular substrate and thus the biological functions associated with the binding. These functions, when related to the ICAM-3 / LFA-1 interaction, may be inhibited by anti-ICAM-3 antibodies.

As a result, monoclonal antibodies capable of binding ICAM-3 can be used as anti-inflammatory agents in human patients and mammals. These agents differ from general anti-inflammatory agents in that they are capable of selective inhibition of adhesion and do not cause side effects, for example, free of renal damaging side effects of conventional agents.

Because ICAM-3, primarily its soluble form, acts as an antibody to the CD18 family, it can be used to inhibit inflammation. In addition, functional derivatives and antagonists of ICAM-3 can be used to inhibit inflammation.

ICAM-3 also mediates in part the adhesion processes necessary for the development of inflammatory reactions, such as delayed-type hypersensitivity reactions. Therefore, antibodies suitable for binding to ICAM-3, preferably monoclonal antibodies, have a therapeutic potential to attenuate and eliminate such reactions.

In addition, ICAM-3 antagonizes ICAM-1 / LFA-1 interaction and therefore ICAM-3, in particular its soluble form and its functional derivatives, can be used to suppress the delayed-type hypersensitivity reaction.

There are two ways to utilize this therapeutic potential. First, a patient with a delayed-type hypersensitivity reaction is administered a composition comprising an anti-ICAM-3 monoclonal antibody or a soluble derivative of ICAM-3. Such formulations are administered, for example, to patients who are exposed to such antigens, such as poison ivy or toxic oak. Alternatively, the patient is administered a monoclonal antibody capable of binding ICAM-3, thereby preventing the development of an inflammatory reaction. Thus, the antigen co-administered with the anti-ICAM-3 antibody may provide the patient with a temporary tolerance to the subsequent presence of the antigen.

Because LFA-1 deficient LAD patients do not develop an inflammatory response, it is expected that antagonists of the natural LFA-1 ligands will also inhibit the inflammatory response. The anti-inflammatory activity of anti-ICAM-3 antibodies forms the basis of therapeutic use in the treatment of chronic inflammatory diseases and autoimmune diseases such as erythematous lupus, autoimmune thyroiditis, experimental allergic cerebrovascular disease (EAE), multiple sclerosis, for the treatment of rheumatoid arthritis. Such antibodies may also be used to treat psoriasis.

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

In accordance with the invention, inflammatory and immune rejection responses can be suppressed, i.e. inhibited or attenuated, by administering to the patient sufficient amounts of an anti-inflammatory agent to suppress inflammation. For example, an anti-inflammatory agent can be used to bind ICAM-3, a fragment thereof, substantially pure ICAM-3, a functional derivative thereof, a non-immunoglobulin antagonist, or a non-immunoglobulin antagonist other than ICAM-3 LFA-1. Particularly preferred as an anti-inflammatory agent is the soluble functional derivative of ICAM-3. As an additional component for the inhibition of inflammation, an antibody capable of binding LFA-1, a non-immunoglobulin antagonist of LFA-1, substantially pure ICAM-1 and / or ICAM-2 or derivatives thereof, or an anti-ICAM-1 and / or anti-ICAM-2 antibody or a fragment thereof.

An additional immunosuppressive agent may be administered as an additional component in the treatment of an inflammatory response of a specific defense system. This agent is generally administered at a sub-optimal dose (suboptimal dose). The suboptimal dose is sufficient because of the synergistic effect of the agents of the invention. Exemplary immunosuppressive agents include dexamethasone, azathioprine, ICAM-1, ICAM-2 or cyclosporin A.

Reactions of the non-specific defense system result in several inflammatory reactions mediated by leukocytes without immunological memory. Examples of such cells include granulocytes and macrophages. In this sense, inflammation is considered to be a consequence of the response of a non-specific defense system when inflammation is caused, mediated, or associated with the reaction of a non-specific defense system. Examples of inflammation at least partially caused by the reaction of a non-specific defense system include inflammations associated with conditions such as: adult respiratory distress syndrome (ARDS), blood poisoning, multiple organ injury syndrome caused by trauma or bleeding, glomerulonephritis or other tissue reperfusion inflammation, dermatosis with acute inflammatory component, acute purulent meningitis, or other inflammatory lesions of the central nervous system such as stroke, heat injury, hemodialysis, leucapheresis, ulcerative colitis, crohn's disease, gonadal ulcerosis, gonorrhagic toxicity.

The anti-inflammatory agents of the present invention are compounds that specifically antagonize the interaction between the CDI8 complex on granulocytes and endothelial cells. Examples of such antagonists include ICAM-3, its functional origin 15

It is a non-immunoglobulin antagonist of ICAM-3 other than ICAM-1 and ICAM-2 or members of the CD18 family.

Because ICAM-3, in particular its soluble form, acts in the same way as an antibody specific for the CDI8 family, it can be used to inhibit organ or tissue rejection caused by cellular adhesion dependent functions. In addition, an anti-ICAM-3 antibody, a functional derivative or antagonist of ICAM-3 can be used to inhibit such rejection.

ICAM-3 and anti-ICAM-3 antibodies can be used to inhibit the rejection of solid organs or tissues, such as kidney, and rejection of non-solid organs, such as bone marrow, and modify autoimmune responses. Importantly, when using monoclonal antibodies capable of recognizing ICAM-3, organ transplantation can also be accomplished in individuals that are different from HLA.

The immune response to therapeutic or diagnostic agents such as bovine insulin, interferon, tissue-type plasminogen activator, or murine monoclonal antibodies significantly weakens the therapeutic or diagnostic value of such agents and can in some cases cause disease such as serum disease. Such situations can be remedied by the antibodies of the invention. To this end, anti-ICAM-3 antibody is administered in combination with said therapeutic or diagnostic agent. The administration of the antibody prevents the body from recognizing the drug being administered, thus preventing the immune response from being triggered. In the absence of an immune response, the patient may be further administered the above therapeutic or diagnostic agent.

For the treatment of the disease, ICAM-3, preferably a soluble form or a functional derivative thereof, may be administered alone or in combination with ICAM-1 or ICAM-2 or an antibody capable of binding LFA-1. Thus, these molecules can be used in soluble form to inhibit rejection of transplanted organs or tissues. To reduce the immunogenic effect of therapeutic or diagnostic agents, the use of ICAM-3 or a functional derivative thereof is carried out in the same manner as anti-ICAM-3 antibodies.

The agents of the invention can also be used to prevent the migration of hematopoietic tumor cells that require CD18 family function for their migration. In doing so, the patient in need of treatment is provided with sufficient amounts of an antibody, a toxin derivative or fragment thereof, or a toxin derivative of the fragment, substantially pure ICAM-3, a functional derivative thereof, or ICAM-2 or ICAM-2 to prevent metastasis. other than an immunoglobulin antagonist.

In another embodiment of the invention, growth of tumor cells expressing ICAM-3 is inhibited. In doing so, the patient is provided with an antibody sufficient to bind ICAM-3, a toxin derivative, a fragment thereof, a toxin derivative of the fragment, a toxin derivative of a CD18 family, or

Toxins of a functional derivative of a CD 18c family molecule are added.

The agents of the invention may also be used to inhibit the growth of tumor cells expressing LFA-1. In this process, the patient is administered sufficient amounts of the ICAM-3 toxin derivative or the functional derivative of the ICAM-3 functional derivative to suppress growth.

The agents of the invention may also be used to prevent HIV infection. An effective amount of an HIV infection prevention agent is administered to an HIV-infected patient. Although the present invention is primarily intended to prevent infection with HIV-1, the method is applicable to any variant of HIV, such as HIV-2, that infects cells in an inhibitory manner with agents of the invention. These variants are considered equivalent to HIV-1 for the purposes of the present invention.

This procedure is based on the recognition that HIV-stimulated expression of LFA-1 and, in some cases, LFA-1 binding ligand, promotes cellular adhesion reactions, which increases the time of contact between infected and healthy cells, transfer from an infected cell to a healthy cell. Therefore, agents that modify LFA / ligand interactions inhibit HIV infection, particularly HIV-1 infection. LFA-1 / ligand interaction inhibitory molecules inhibit HIV infection in many ways. One possible way is to attenuate the binding of the LFA-1 ligand expressed by the HIV-infected cell to the CD11 / CD18 receptor of a healthy T cell. Alternatively, they can attenuate the binding of CDI 1 / CD18 receptors expressed by HIV infected cells to LFA-1 produced by healthy T cells. ICAM-3, a fragment, functional derivative thereof, or anti-ICAM-3 antibody may be used to attenuate binding to the CD11a / CD18 receptor or the LFA-1 binding ligand.

The agent of the invention is administered to the treated patient in an amount sufficient to inhibit HIV infection. This means that the medicine will reduce or prevent HIV infection, depending on the dose and the route of administration. The agent is administered to an HIV-infected patient or a patient exposed to such an infection.

The agents of the invention may be administered for prophylactic or therapeutic purposes. In the case of prophylactic administration, the agent is administered prior to, or shortly after, symptoms of viral infection, for example, prior to the onset of any symptom of infection. Prophylactic administration of the agent of the invention prevents or attenuates the development of HIV infection. For therapeutic use, the agent is administered at or shortly after detection of virus-infected cells. Therapeutic use is intended to attenuate further HIV infection.

Thus, the agents of the invention may be used prior to the onset of a viral infection, to attenuate a putative HIV infection, or after the practical detection of virus-infected cells to attenuate further infection.

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The agents of the invention may also be used to treat AIDS diseases and to prevent HIV infection, particularly HIV-1 infection, by co-administering the following components:

I. ICAM-3, a soluble form thereof, CD11, preferably CD11a, CD11b or CD111, a soluble derivative thereof, CD18, a soluble derivative thereof, or a CD111 / CD18 heterodimer or a soluble derivative thereof, and / or

II. Antibody capable of binding ICAM-3,

III. A cell or particle associated with CD4 or a soluble derivative thereof, and / or

ARC. A molecule capable of binding CD4, preferably an antibody or antibody fragment.

The present invention encompasses inhibiting the migration of HIV-infected cells. An effective amount of an anti-migration agent is administered to an HIV-infected patient.

Examples of anti-migration agents used in the present invention include ICAM-3, a fragment or functional derivative thereof, and anti-ICAM-3 antibodies which inhibit the binding of HIV-infected T cells to LFA-1 ligand. The antibody capable of binding ICAM-3 inhibits migration by weakening the binding of ICAM-3 expressed by HIV-infected T cells to cells expressing the CD11 / CD18 receptor. For example, an antibody capable of binding ICAM-3 may be used to inhibit the binding of a cell to the CD11a / CD18 receptor.

The agent of the invention is administered to the patient in an amount that inhibits the migration of HIV-infected or other virus-infected T cells. This means that the amount used is sufficient to attenuate or prevent T-cell migration.

These agents may be administered prophylactically or for therapeutic purposes. For prophylactic use, the agent is administered before the onset of symptoms of a viral infection, such as before, during or shortly after infection, but prior to the onset of any symptoms of infection. The purpose of prophylactic use is to attenuate or prevent the migration of virally infected T cells. For therapeutic use, the agent is administered at or shortly after detection of virally infected T cells. Therapeutic use is intended to attenuate further migration of such T cells.

Thus, the agents of the invention may be used prior to the onset of viral infection, for example, to inhibit putative migration of infected T cells or after the practical detection of virus-infected cells.

In another embodiment of the present invention, an agent for modulating the LFA-1 / ICAM-3 interaction is used to treat asthma. An effective amount of an anti-asthma agent is administered to the patient.

Examples of anti-asthma agents used in the present invention include ICAM-3, a fragment or functional derivative thereof, and anti-ICAM-3 antibodies which are capable of inhibiting the binding of a cell to LFA-1. Antibodies capable of binding ICAM-3 inhibit eosinophilic migration by attenuating the binding of cell-expressed ICAM-3 to cells expressing the CD11 / CD18 receptor.

The anti-asthma agent of the present invention is administered to the patient in an amount which is attenuating or inhibiting the severity, severity and duration of the symptoms of asthma.

The agents of the invention may be administered alone or in combination with one or more known anti-asthma agents. Examples of these include methylxanthines such as theophylline, β-adrenergic agonists such as catecholamine, resorcinol, saligenin and ephedrine, glucocorticoids such as hydrocortisone, chromones such as chromoline sodium and anticholinergics such as atropine. The combination makes it possible to reduce the amount of known anti-asthma agents required.

The agents of the invention may be administered prophylactically or for therapeutic purposes. In the case of prophylactic use, the drug is administered before the symptoms of asthma appear. The purpose of prophylactic application is to attenuate or prevent a possible asthmatic response. For therapeutic use, the agent is administered at or shortly after the onset of symptoms of asthma. The aim of the therapeutic application is to attenuate the current period of asthma. Thus, the agents of the invention may be administered prior to the onset of a putative asthmatic period (to attenuate the severity, duration and extent of the putative period) or after the onset of the period.

A monoclonal antibody capable of binding ICAM-3 can also be used to map and visualize the expression of ICAM-3 and the site of inflammation. In doing so, the anti-ICAM-3 monoclonal antibody is detectably labeled, for example, using a radioisotope, an affinity signal such as biotin or avidin, a fluorescent signal, or a paramagnetic atom. This type of marking is carried out in the usual manner. The labeled antibody can then be used for diagnostic imaging. The clinical application of diagnostic imaging antibodies is described by Grossman Η. B. Urol. Clin. North America 13, 465-474 (1986); Unger, E.C., et al., Invest. On radio. 20: 693-700 (1985) and B.A. Khaw et al., Science 209: 295-297 (1980).

The expression of ICAM-3 can also be detected by a hybridization assay. Examples of hybridization probes include mRNA, cDNA, genomic DNA or synthetic oligonucleotide probes that bind to the ICAM-3 gene sequence or the ICAM-3 mRNA sequence present in a cell capable of expressing ICAM-3. The hybridization assay can be carried out in a conventional manner (Maniatis T. et al., Molecular Cloning, Laboratory Manual, Coldspring Harbor, New York, USA, 1982); Haymes B.D. et al., Nucleic Acid Hybridization, Practical Approach, IRL Press, Washington, DC, USA (1985).

Detection of the site of expression of ICAM-3 with a labeled antibody or nucleic acid probe can be used to diagnose tumor development. The diagnosis can be made by removing the patient

A blood or blood sample is taken and incubated in the presence of a detectably labeled or labeled antibody. The process is preferably carried out in a non-destructive manner, by magnetic imaging or by fluorography. This diagnosis can be used, for example, in an organ transplant patient, for example, to detect early signs of a potential rejection after a kidney transplant. This assay may also be used to detect a predisposition to rheumatoid arthritis or other chronic inflammatory disease.

By radiolabeling the anti-ICAM-3 antibody or antibody fragment, the antigen can be detected by radioimmunoassay. The radioimmunoassay (RIA) assay is carried out in the usual manner (Work, T.S., et al., 1978, Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company). The fluorescent compound, enzyme or any other conventional labeling method may also be used to label the antibody.

In addition to localizing the site of inflammation, the antibody that binds ICAM-3 can be used to test for biological fluids that detect circulating ICAM-3. The assay may be performed by any conventional method for assaying biological fluids. If ICAM-3 is detected in the biological fluid, it indicates the presence of an inflammatory response or other biological function mediated by ICAM-3. In addition, the presence of ICAM-3 in amniotic fluid indicates a complication during pregnancy. Any biological fluid may be used for the assay. However, blood, serum, plasma, synovial fluid, amniotic fluid, spinal fluid or urine are preferred.

To induce a therapeutic effect of ICAM-3, the patient is administered the entire ICAM-3 molecule or any therapeutically effective peptide fragment thereof. Particularly preferred are the therapeutically effective soluble peptide fragments of ICAM-3.

ICAM-3 and its functional derivatives may be synthetically produced by recombinant DNA technology, proteolysis, or a combination thereof. The therapeutic effect of ICAM-3 can be enhanced by the use of functional derivatives containing additional amino acid residues to enhance binding or activity of ICAM-3 to a carrier. Also included are functional derivatives of ICAM-3 which lack certain amino acid residues or which contain altered amino acid residues, provided that such derivatives retain the biological or pharmacological action of ICAM-3.

The antibodies of the invention and the ICAM-3 molecule are considered to be substantially free of natural impurities if the composition containing them is substantially free of the substances with which these products are commonly found in nature.

The present invention encompasses antibodies, biologically active fragments thereof, which may be polyclonal or monoclonal and which are capable of binding ICAM-3. These antibodies can be produced in an animal body, tissue culture or by recombinant DNA technology.

ICAM-3 and its derivatives may be administered alone or in combination with ICAM-1 and / or ICAM-2. The anti-ICAM-3 antibody or other molecules capable of binding ICAM-3 or molecules derived from ICAM-3 may be administered alone or in combination with an anti-ICAM-1 antibody and an anti-ICAM-2 antibody. When administering the antibody or ICAM-3 antibody fragment or ICAM-3 or a fragment, variant or derivative thereof, the dosage employed will depend on various factors. These include the patient's age, weight, height, sex, general health, and previous medical conditions. The required dose of antibody will generally range from about 1 pg / kg to about 10 mg / kg body weight, but lower or higher doses may be used. When the patient is administered ICAM-3 or a functional derivative thereof, the dosage will generally range from about 1 pg / kg to about 10 mg / kg body weight, but lower or higher doses may be used. As discussed in more detail below, a therapeutically effective dose of an anti-ICAM-3 antibody can be reduced by co-administration of an antiLFA-1 antibody, an anti-ICAM-1 antibody, and an anti-ICAM-2 antibody. By co-administration, it is understood that the two agents are administered at such short intervals that both can be detected simultaneously in the patient's serum.

Both the antibody that binds ICAM-3 and ICAM-3 itself can be administered intravenously, intramuscularly, subcutaneously, enterally, topically or parenterally. In the case of injection of the antibody or ICAM-3, injection may be effected by continuous injection, single injection or multiple injection.

The composition of the invention is administered to a patient in a physiologically effective amount. An amount is considered physiologically effective when the dosage and mode of administration of the composition are sufficient to attenuate or prevent the physiological effect dependent on ICAM-3. For example, when the composition of the invention is administered to a patient for anti-inflammatory purposes, it is considered physiologically effective if the dose used suppresses the inflammation.

In addition, anti-ICAM-3 antibodies, or fragments thereof, may be administered alone or in combination with one or more other immunosuppressive agents, particularly in tissue or organ transplant patients. The administration of such agents may be prophylactic or therapeutic. For prophylactic use, the immunosuppressive agent is administered prior to, during, or shortly after the onset of an inflammatory response or symptom, such as organ or tissue transplantation, but prior to the onset of any symptom of organ rejection. The purpose of prophylactic administration of active ingredients is to prevent or attenuate the rejection of any inflammatory response, such as a transplanted organ or tissue. For therapeutic use, the active compounds are administered at or shortly after the onset of symptoms of inflammation such as rejection of an organ or tissue. The therapeutic application is directed to an inflammatory response such as a transplanted solid organ or tissue such as a kidney,

Breast rejection of non-solid or non-solid organs such as bone marrow.

The anti-inflammatory compositions of the present invention are therefore administered prior to the onset of inflammation, i.e. to attenuate the putative inflammation or after the initiation of the inflammatory process.

A formulation is considered pharmacologically acceptable if its administration is tolerated by the patient being treated. Such a composition is administered in a therapeutically effective amount, which means that the amount administered is physiologically significant. A substance is considered physiologically significant if its presence results in a detectable change in the physiological processes of the patient.

The antibodies and ICAM-3 molecules of the invention are formulated according to conventional methods for the preparation of pharmaceutical compositions. The above materials or functional derivatives thereof are admixed with a pharmacologically acceptable carrier. Carriers and types of formulations that may be used with other human proteins, such as human serum albumin, may be found in Remington's Pharmaceutical Sciences [16]. Osol A., Mack, Easton PA, USA (1980)]. An effective amount of the active ingredient of the present invention is admixed with an appropriate amount of a carrier to produce an effective dosage form and a pharmaceutically acceptable formulation. In addition, the antibody of the invention can be humanized, for example, by chimerization or CDR vaccination to improve its tolerance. The chimerization of antibodies, in particular anti-ICAM-1 antibodies, is described, for example, in British Patent Nos. 9009548.0 and 9009549.8, and International Patent Applications PCT / US91 / 02942 and PCT / US91 / 02946.

The duration of action may be influenced by other pharmacological methods. Controlled release formulations may be prepared using polymers that complex or absorb the active compound of the present invention. The extent and duration of controlled release can be controlled within a limited range by selecting an appropriate macromolecular matrix, varying the concentration of macromolecules employed, and coatings. Another method for controlling the duration of action is to formulate the active ingredient of the present invention in particulate form with polymeric materials such as polyesters, polyamino acids, hydrogels, polylactonic acid or ethylene-vinyl acetate copolymer. As an alternative to incorporation into polymeric particles, the active ingredient may be microencapsulated, for example, by coacervation technique or surface contact polymerization, gelatin or poly (methylmethacrylate), microencapsulation, colloidal systems such as liposomes, microspheres, with sustained release systems.

One embodiment of the pharmaceutical compositions of the present invention

(a) an anti-inflammatory agent such as an antibody that binds ICAM-3, a fragment thereof, ICAM-3, a functional derivative thereof, or a non-immunoglobulin antagonist of ICAM-3 other than ICAM-1 and ICAM-2, and

b) at least one immunosuppressive agent.

Examples of immunosuppressive agents include dexamethasone, azathioprine or cyclosporin A.

The present invention will be further illustrated by the following examples, without limiting the scope thereof to the examples.

Example 1

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

LFA-1 coated plates develop adhesion cell lines and lymphocytes as described above (Dustin et al., Cold Spring Harbor Symp. Quant. Biol. 54: 753-765 (1989)]. LFA-1 purified from JY lysate was absorbed in a 96-well microtiter plate (Linbrotiters) at 1100 sites / μτη ² . The nonspecific binding sites were blocked with 1% BSA and washed with assay medium (PBS, 5% FBS, 2 mM MgCl ₂ and 0.5% HSA). For specific inhibition of LFA-1, the microtiter plate is incubated for 30 minutes at room temperature with 1/200 dilution of TS 1/22 (anti-LFA-alpha) typhoid. Resting T-cells were isolated from whole blood by adherence to plastic and filtration on a nylon filter, yielding 91% CD2 +. Cells are cultured in the presence of 10 pg / ml phytohemagglutinin (PHA) to generate PHA blasts. Cells were labeled with fluorochrome BCECF (Molecular Probes Inc.) and washed and resuspended in assay medium. For MAb pretreatment, cells were incubated with 1/200 dilution of typhoid fever for 45 minutes at 4 ° C and 105 cells per well were weighed. The cell line was treated with solid phase LFA-1 for 1 hour at 37 ° C, and unbound cells were removed by plating into 6 size 23 needles, while lymphocytes were harvested at 30 g for 5 minutes and at 37 ° C. incubate for one minute. To remove unbound lymphocytes, the plates were washed eight times with 100-fold excess floating medium. Floatation completely removes unbound T-lymphocytes, which are quite complicated due to their small size. Fluorescence measurement was performed directly on a 96-well plate using a fluorescence concentration analyzer (Baxte). The MAb was used at saturation concentration (1/200 dilution in rash) and TS 1/22 (anti-CDIIa, IgGl) [Sanchez-Madrid et al., Proc. Natl. Acad. Sci. USA 79, 7489-7493 (1982)], RR1 / 1 (anti-ICAM-1, IgG1) [Rothlein et al., J. Immunol. 137, 1270-1274 (1986)], CBR-IC2 / 2 (antiICAM-1, IgG2a) (deFougerolles et al., J. Exp. Med. (1991)), and CBR-IC3 / 1 (anti-ICAM-3). IgG1). For the production of CBR-IC3 / 1, P3X63Ag8.653 mouse myeloma from SKW3-treated Balb / c mice [Gefter et al., Som. Cell Gene. 3

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231-236 (1977)] and tested on 600 hybridomas for ability to inhibit SKW3 binding to purified LFA-1. CRB-IC3 / 1 does not react with purified LFA-1 or with COS cells transfected with ICAM-1, ICAM-2 or LFA-1 cDNA. The assay is performed in four replicates and the standard deviation is determined.

Example 2

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

Known cell lines (see Table 2) are used for this assay [Hibbs et al., J. Clin. Invest. 85: 674-681 (1990). Dextran sedimentation and Ficoll-Hypaque (1077) centrifugation [Dustin et al., J. Cell. Biol. 107: 321-331 (1988)] to produce peripheral blood mononuclear cells (PBMCs). Neutrophil cells were harvested from the cell pellet and contaminating erythrocytes were removed by hypotonic digestion. Lymphocytes and monocytes were separated by cytometric analysis based on forward and vertical light scattering, and the result was monitored with monocytes and T cell-specific MAb. Based on adhesion from PMBC to plastic, lymphocytes are enriched and cells are cultured in the presence of 10 pg / ml phytohemagglutinin (PHA). The sample was analyzed on an EPICS Type V Flow Cytometer, calibrated with an EPICS Immune-Brite Fluorescence bead (Coulter). ICAM-3 expression on resting lymphocytes is two to three times higher than that of CD3 or LFA-1, while monocytes express three to four times more ICAM-3 than LFA-1. In neutrophils the level of ICAM-3 expression is the same as that of Mac-1 (CD11b / CD18). ICAM-3 linked to ΡΙ could not be detected by treating cells with phospholipase C.

Table 2

Relative surface antigen expression in ICAM by immunofluorescence flow cytometric analysis

Nucleus Type Specific linear fluorescence intensity (*) of ICAM-1 (RR1 / 1) the ICAM-2 (CBR-IC2 / 1) of ICAM-3 (CBR-IC3 / 1) static lymphocytes 4 22 106 1 day PHA blast 22 18 78 3 days PHA blast 85 42 205 5 days PHA blast 25 45 223 monocyte 13 39 224 Neutrophil cell 1 0 213 T lymphoblastoid SKW3 0 98 73

Nucleus Type Specific linear fluorescence intensity (*) of ICAM-1 (RR1 / 1) the ICAM-2 (CBR-IC2 / 1) of ICAM-3 (CBR-IC3 / 1) Jurkat 2 166 161 Sup T 10 113 19 Molt 4 1 242 117 B lymphoblastoid JY 154 119 47 SLA 224 113 306 Ramos 132 136 112 Raji 266 88 0 monocyte U937 62 67 37 HL60 17 43 203 Melanoma BK 896 3 0 RPMI 7591  475 ⁰ 0 erythroleukemia K562 293 143 0 Mixed (**) HUVEC 31 494 0 Hep G2 1526 41 0 HeLa 1082 0 RD3 / 5 0 9 0 FS1,2,3 409 0 0 A-172 0 0 0

*: Membrane expression is performed by immunofluorescence flow cytometric analysis as described. The values given are at least two replicates. Fluorescent beads were used to calibrate the cytomctcr, and one unit corresponds to about 103 fluorescent equivalents (Coultc Diagnostics, Hialcah, FL).

**: Mixed cell lines include: human umbilical vein endothelial cell (HUVEC), human breast carcinoma (Hcp G2), human squamous cell carcinoma cell line (HeLa), human rhabdomyosarcoma (RD3 / 5), human fibrosarcoma (FS1,2,3) ) cs human glioblastoma (A-172).

Example 3

Immunoprecipitation assay of ICAM-3

Cells surface-labeled h ^l25 I using iodogen [Kishimoto, et al, J. Biol. Chem. 264: 3588-3595 (1989)]. Cells were disrupted with 1% Triton X-100. The lysate was treated with Sepharose-loaded calf IgG and incubated with Sepharose-bound MAb for 2 hours. The beads were washed and boiled in sample buffer containing 50 mM Tris, 1% SDS, and 1% 2-mercaptoethanol. Unreduced samples were boiled in 2-mercaptoethanol-free buffer and treated with 20 mM iodoacetamide. Samples were analyzed on a 7% vertical polyacrylamide gel sheet (Laemmli UK: Natura 227: 680-685 (1970)).

And the proteins were autoradiographically visualized. Samples were treated with N-glycanase (g enzyme) as described above (Tarentino et al., Biochemistry, 24, 4665-4671 (1985)) under predetermined conditions (10 units / ml, 37 ° C, 18 hours) optimizing the cleavage of N-linked oligosaccharides from the peptide backbone. MHC class I contains N-linked carbohydrates, whereas ICAM-2 (m, 60,000, 6 N-linked sites, base chain MR, 28383) is highly glycosylated.

Example 4

Distribution of ICAM-3

The anti-ICAM-3 antibodies of the invention are used to test the distribution of ICAM-3 protein in various tissues. The assay is performed by flowing cytometric analysis. Table 3 lists cell types showing surface expression of ICAM-3. The results indicate that expression of ICAM-3 is limited to haemopoietic cells. Flow cytometric data were verified by immunohistochemical staining of tissue samples using labeled anti-ICAM-3 antibody. In accordance with flowing cytometric data, immunohistochemical studies indicate that expression of ICAM-3 is limited to haemopoietic cells.

Table 3

Relative surface antigen expression in ICAM as measured by immunofluorescence flow cytometric analysis

Nucleus Type Specific Linear Fluorescence Intensity (*) of ICAM-1 (RR1 / I) the ICAM-2 (CBR-IC2 / 1) of ICAM-3 (CBR-1C3 / 1) static lymphocytes 4 22 106 1 day PHA blast 22 18 78 3 days PHA blast 85 42 205 5 days PHA blast 25 45 223 monocyte 13 39 224 Neutrophil cell 1 0 213 T lymphoblastoid SKW3 0 98 273 Jurkat 2 166 161 Sup T 10 113 19 Molt 4 1 242 117 B lymphoblastoid JY 154 119 47 SLA 224 113 308

Scjtmagtípus Specific Linear Fluorescence Intensity (*) of ICAM-1 (RR1 / 1) the ICAM-2 (CBR-IC2 / 1) of ICAM-3 (CBR-IC3 / 1) Ramos 132 136 112 Raji 266 88 0 monocyte U937 62 67 37 HL60 17 43 203 Melanoma BK 896 3 0 RPMI 7591 475 0 0 erythroleukemia K562 293 143 0 Mixed(**) HUVEC 31 494 0 HepG2 1526 41 0 HeLa 1082 0 0 RD3 / 5 0 9 0 FSI, 2.3 409 0 0 A-172 0 0 0

*: Membrane expression is performed by immunofluorescence flow cytometric analysis as described. The values given are the results of at least two replicates. Fluorescent beads were used to calibrate the cytometer and one unit corresponds to about 103 fluorescent equivalents (Coulte Diagnostics, Hialeah, FL). * ·: The mixed cell lines include: human umbilical cord endothelial cell (HUVEC), human mcllc carcinoma (ffep G2), human cytoplasmic carcinoma cell line (HeLa), human rhabdomyosarcoma (RD3 / 5), human fibrosarcoma (FS 1.2, 3) and human glioblastoma (A-172).

Example 5

Purification of ICAM-3

ICAM-3 was purified by immunoaffinity chromatography (deFougerolles et al., J. Exp. Med. 179, 619-629 (1994)), in which the antiICAM-3 antibody was immobilized on the matrix in the usual manner. Various starting materials are used to prepare ICAM-3. Examples include SKW3, human thymus tumor cell line (ATCC TIB215) and any human almond. Purification of ICAM-3 is carried out according to known procedures (deFougerolles et al., 1992, J. Exp. Med. 175: 185-190). Wash and elution is performed according to the antigen to be purified and the antibody used in immunoaffinity chromatography.

ICAM-3 purified from SKW3 cells or human tonsil cells has a molecular weight of about 120-124 kD, whereas ICAM-3 purified from neutrophil cell lysate

EN 217 176 B shows a wide band of about 120-150 kD.

ICAM-3 purified in this manner retains the ability of anti-ICAM-3 antibodies to bind to LFA-1 on various cells (Figures 6.5 and 7).

Example 6

Identification of ICAM-3 of different molecular weights

The molecular weight of ICAM-3 expressed on different cell types was determined by immunoprecipitation assay and polyacrylamide gel electrophoresis. The molecular weight of ICAM-3 precipitated from neutrophils is different from that of ICAM-3 obtained on lymphocytes. The molar mass of ICAM-3 isolated from lymphocytes and lymphoid cell lines is approximately 120-124 kD. The molecular weight of ICAM-3 isolated from neutrophils is slightly higher than that of lymphoid cells and forms a diffuse band at 120-150 kD (Figure 4).

Such size variability is not uncommon for glycoproteins of the ICAM-3 family. ICAM-1 shows similar molecular weight changes for different cell types. The change in molecular weight for ICAM-1 results from different degrees of glycosylation. Therefore, the molecular weight change observed with ICAM-3 is also likely to be explained by differences in glycosylation. This can be easily verified by treating ICAM-3 isolated from neutrophil cells with different glycosidases and measuring its effect on molecular weight change.

The difference in glycosylation with ICAM-1 affects the binding of Mac-1 (CD11b / CD18). Therefore, by altering the glycosylation of ICAM-3, ICAM-3 derivatives can be prepared which have a modified affinity for various ICAM-3 ligands, such as the CD11 / CD18 family of glycoproteins.

Example 7

ICAM-3 antibodies

Mice are injected with a formulation consisting of purified ICAM-3 protein and vehicle from SKW3 cells or tonsil cells. Immunized animals may be prepared by conventional means (e.g. Harlow 10 et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988) or Yelton et al., Annu. Port. Biochem. 50: 657-680 (1981)].

The various anti-ICAM-3 antibodies obtained are shown in Table 4. Each antibody is identified by its reactivity towards purified, immobilized ICAM-3 and verified by ELISA. Positive MAb assays on ICAM-3 positive cell lines were performed and immunoprecipitated analysis was performed on radiolabeled ICAM-3 positive cell lysate. The MAb is also assayed for PMA-induced inhibition of SKW3 cell aggregation in the presence of anti-ICAM-1 and anti-ICAM-2 antibodies. Alternatively, the anti-ICAM-3 antibody can be identified by inhibiting the binding of SKW3 cells to immobilized purified ICAM-3.

One of the evidences of the existence of the third LFA-1 ligand is that the agg30 region of PMA-stimulated SKW3 cells also has an ICAM-1 / ICAM-2 independent pathway. The present invention enables the inhibition of this aggregation using a mouse anti-ICAM-3 polyclonal antiserum or a combination of CBR-IC3 / 1 and CBR-IC3 / 2.

Table 4 ICAM-3mAb

mAb isotype PMA induction * aggregation itself + + ICl 2mAb + IC1 + 2 + IC3 / 1 mAb CBR-IC3 / 2 IgG2a, k 3 2 0 CBR-IC3 / 3 IgG2a, k 4 3 2 CBR-IC3 / 4 IgM, k 4 3 2-3 CBR-IC3 / 5 IgG2a, k 2 2 0-1 CBR-IC3 / 6 N.D. 5 5 5 ICAM-3 antiserum 3 0 0 CBR-IC3 / 1 IgG, k 5 4 - HP2 / 19 ** 1

*: The degree of aggregation is performed according to J. Exp. Med. 1991, 0 denotes lack of aggregation **: Another ICAM-3 mAb from F. Sanchez-Madrid, mAb not further characterized after results, presumably ICAM-third

CBR-IC3 / 2, 3/3, 3/5 immunoprecipitative assay for CBR-IC3 / 1. The mAb yields the same 120 kD band after Westemlata and performs 60 spot analysis.

HU 217 176 B

Example 8

Immunoassay with anti-ICAM-3 antibody

Anti-ICAM-1 antibody (RR1 / 1), anti-ICAM-2 antibody (CBRI-C2 / 2) and anti-ICAM-3 antibody (CBR-IC3 / 1 and CBR-IC3 / 2 combination) block the following processes:

1. Adhesion of PMA-stimulated SKW3 cells to purified ICAM,

2. Division of phytohemagglutinin (PHA) stimulated cells and

3. mixed lymphocyte response (MLR).

PMA has previously been shown to activate LFA-1, thereby increasing the binding of ICAM-3 (Dustin et al., 1989, Natur. 341, 619). Figure 5 shows a similar activation mechanism for LFA1 / ICAM-3 binding.

Various antibodies were tested for their ability to inhibit the binding of PMA-stimulated SKW3 cells to purified ICAM-1 and ICAM-3 (Figure 6). CBR-IC3 / 1 and CBR-IC3 / 2 antibodies, when administered alone, do not effectively inhibit the binding of PMA-stimulated SKW3 cells to purified ICAM-3. However, the same binding can be effectively blocked by a combination of the above two antibodies. CBR-IC3 / 1 monoclonal antibody alone inhibits the binding of ICAM-3 to purified LFA-1 (deFouregolles: J. Exp. Med., Cited above).

By further examining the binding of PMA-stimulated SKW3 cells to ICAM-3, it is determined whether the process is dependent on temperature and the presence of cations. Like ICAM-1, ICAM-3 / LFA-1 binding is dependent on both temperature (Figure 8) and the presence of cations.

Figure 9 shows the effect of different antibodies on PHA-stimulated cell division. Known Krensky et al., J. Immunol. 131: 611-616 (1983)] that PHA stimulates cell proliferation by inhibiting anti-LFA-1 in anti-ICAM-1 antibodies. Anti-ICAM-2 antibodies or combinations of anti-ICAM-1 and anti-ICAM-2 antibodies have less effect on PHA-stimulated cell division. However, it is

1. anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3 antibodies,

2. two anti-ICAM-3 antibodies (IC3 / 1 and IC3 / 2) and

3. The combination of anti-ICAM-1 and anti-ICAM-3 antibodies effectively inhibits PHA-stimulated cell division.

Example 9

Mixed lymphocytic reaction

The MLR assay measures the reactivity of immunology. During the assay, chemically fixed foreign cells (stimulator cells) are added to a solution containing PBL from different individuals (responder cells) and the level of response is measured. As an index, proliferator cell proliferation is used [Krensky et al., J. Immunol. 131: 611-616 (1983)]. This assay is the first step in identifying formulations for treating graft rejection.

The effect of different antibodies and antibody combinations on the MLR reaction is summarized in Figure 10. Briefly, anti-ICAM-3 alone has little effect. A greater blocking effect can be achieved by combining antiICAM-1 and anti-ICAM-3 antibody. The greatest response is obtained by combining anti-ICAM-1, anti-ICAM-2, and anti-ICAM-3 antibodies.

Example 10

Sequence of ICAM-3 peptide

ICAM-3 is purified from human tonsil cells by immunoaffinity chromatography as described in Example 5. Purified ICAM-3 was digested with Lys-C as usual and the peptide fragments were resolved by HPLC. Gas chromatographic analysis after normal digestion yielded various protein peaks. is shown. Peak 10 and 17 correspond to a peptide fragment of size and structure suitable for sequencing (hereinafter referred to as NK-10 and NK-17 peptides).

The amino acid sequences of the NK-17 and NK-10 peptides were determined by the usual procedure. The protein sequence of NK-10 is KIDRATCPQHLK (SEQ ID NO: 1). The first lysine residue (K) in the sequence is hypothesized because Lys-C cleaves proteins after the lysine residue.

The peptide of NK-17 is KIALETSLSK (SEQ ID NO: 2). Like the NK-10 protein, the first lysine residue is put forward and confirmed by subsequent DNA sequence analysis.

The sequence of NK-10 and NK-17 proteins has a high degree of homology compared to the known sequences of ICAM-1 and ICAM-2. The NK-17 peptide exhibits significant homology with the sequence found in the first Ig domain of ICAM-2. The NK-10 peptide shows poor homology with the sequence in the quad domain of ICAM-1.

Example 11

Cloning of ICAM-3 cDNA

Based on the resulting peptide sequences, degenerate oligonucleotide probes are prepared. Preferred codons known from the nucleotide sequence of ICAM-1 and ICAM-2 are used for probes. The probes obtained from the amino acid sequence of NK-17 and NK-10 have the following nucleotide sequence:

G

G A A T '-AAN-GAN-GTC-TCC-AGG-GCT-ATC-TT-3' (SEQ ID NO: 8)

NK-17 probe, oligo 23mer, carrying 2n (inosine) 24-fold degeneration.

GC A '-TTN-AGA-TGT-TGN-GGG-C AN-GIN-GCN-C-3' (SEQ ID NO: 4) NK-10 probe, 25mer, carrying eight fold degeneration of 5n (iosine).

A cDNA expression library was prepared from almond RNA [Wong et al., Proc. Natl. Acad. Sci. USA 82: 7711-7716 (1985)]. The library was assayed with the NK-17 probe and plaques showing positive hybridization were rescreened with the NK-10 probe. One of the clones, 11.2

Clone HU 217,176 shows an insert of approximately 1.6-1.8 kb. A graphical representation of clone 11.2 and the location of the NK-10 and NK-17 peptides is shown in Figure 12.

The ends of the insert were sequenced in the usual manner using the primer from the connecting vector. The NK-10 probe was used as an internal sequencing primer. The resulting sequence is single-stranded. One skilled in the art will recognize that such a sequence can be verified by routine experimentation.

The resulting sequences are shown in Figure 18. In contrast, we make the following remarks. First, the identified sequence exhibits a high degree of homology to the transmembrane region of ICAM-1. Therefore, all sequences starting after the transmembrane region are deleted (Figure 12) to produce a soluble fragment of ICAM-3. Secondly, the sequence obtained from the 5 'end of clone 11.2 is similar to the 5' end of the naturally occurring gene, based on the sequence homology between ICAM-1, ICAM-2 and ICAM-3.

Example 12

Sequence homology between different ICAM-3 molecules

The nucleotide and amino acid sequences of the NK-10 and NK-17 peptides and the above nucleotide sequences are compared with the known ICAM-1 and ICAM-2 sequences (Figures 13-17).

The results indicate that ICAM-3 exhibits a high degree of homology to both ICAM-1 and ICAM-2. Sequence homologies between ICAM-1 and ICAM-3 are found in the first (Figure 16) and fourth / fifth domains of ICAM-1, as well as in the transmembrane domain of ICAM-1 and some of its cytoplasmic domains (Figures 13 and 14). Further examination of the gene bank database revealed no further identity or similarity to the sequence obtained at ICAM-3. Significant homology is also seen between ICAM-3 and the first domain of ICAM-2 (Figure 17).

Further examination of clone 11.2 provides newer clones that carry a cDNA insert of about 2 to about 2.4 kD. These are most likely full-length cDNA clones, since no larger size has been identified so far.

sequence Listing

2. Information on SEQ ID NO: 1:

(i) sequence characteristics:

(A) length: 152 base pairs (B) type: nucleic acid (C) strand: double-stranded (D) topology: linear (ii) molecule type: DNA (ix) characteristic:

(A) Name / Key: CDS (B) Location: 2-152 (xi) Sequence Description: SEQ ID NO: 1

G GAG TTC CTT TTG CGG GTG GAG Glu Phe Leu Leu Arg Val Glu

1 5 GCT GGA GGG TCC CTG TTT GTG AAC Al a Gly Gly Ser Leu Phe Va 1 As n 1 20 TCT GAG AAA ATC GCC TTG GAG ACG Ser Glu Ly s I 1 e Al a Leu Glu Thr 30 35 GCC AGT GGC ATG GGC TGG G 152 Al a Ser Gly Me t Gly Trp 45 50

CCC CAG AAC CCT GTG CTC TCT Pro Gin Asn Pro Val Leu Ser

10 TGC AGT ACT DAM TGT CCC AGC Cy s Ser Thr 25 As p Cy s Pro Ser TCC CTA TCA AAG GAG CTG GTG Ser Leu Ser 40 Ly s Glu Leu Va 1

2. Information on SEQ ID NO: 2:

(i) sequence characteristics:

(A) Length: 50 amino acids Type 45 (B): Amino acid

Glu 1 Phe Leu Leu Arg 5 Va 1 Glu Pro Gly Gly Ser Leu Phe 20 Va 1 As n Cys Glu Ser Ly s Gly I le Me t Al a Gly Leu 35 Trp Glu Thr Ser

(D) topology: linear (ii) molecule type: protein (xi) Sequence Description: Figure 2. sequences cia Gin Asn Pro Val Leu Ser Al a 10 1 5 Ser Thr Asp Cys Pro Ser Ser 25 30 Leu Ser Lys Glu Leu Val Al a 40 45

2. Information on SEQ ID NO: 3: (i) Sequence Characteristics:

(A) Length: 189 base pairs (B) Type: Nucleic acid (C) Fiber: Double-stranded (D) Topology: Linear (ii) Molecule type: DNA (ix) Characteristics:

EN 217 176 B (A) Name / Key: CDS (xi) Sequence Description: SEQ ID NO: 3 (B) Location: 2-189

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

Pro Leu Arg Gly Ser Thr Val Thr Val Ser Cys Me t Alá Gly

1 5 10 GCT CGA GTC CAG GTC ACG CTG GAC GGA GTT CCG GCC GCG GCC CCG A1 a Arg Va 1 Gin With Thr Leu As p Gly Va 1 Pro A1 a A1 a A1 a Pro 15 20 25 GGG CAG CCA GCT CAA CTT CAG CTA AAT GCT ACC GAG AGT GAC GAC Gly G1 n Pro A1 a Gin Leu Gin Leu Asn A1 a Thr Glu Ser asp As p 30 35 40 GGA CGC AGC TTC TTC TGC AGT GCC ACT CTC GAG GTG CAC GGC CAG Gly Arg Ser Phe Phe Cys Ser A1 a Thr Leu Glu With Hi s Gly Gin 45 50 55 TTC TTG CAG BRANCH 189 Phe Leu Gin 60

2. SEQ ID NO: 4: 20 (i) Sequence Characteristics:

(A) Length: 62 amino acids (B): amino acid (D) topology: linear (ii) molecule type: protein (xi) Sequence description: SEQ ID NO: 4

Pro 1 Leu Arg Gly Ser 5 Thr Va 1 A1 a 15 Arg With Gin With Thr 20 Leu Pro Gly 30 Gin Pro A1 a Gin Leu 35 As p As p Gly 45 Arg Ser Phe Phe Hi s Gly Gin Phe 60 Leu Gin

Thr Va 1 Ser Cys Me t A1 the G1 y 10 asp G1 y Va 1 Pro A1 a A1 the A1 the 25 Gin Juice u Asn A1 a Thr G1 u Neither r 40 Cys Neither r A1 a Thr Leu G1 u Va 1 50 5 5

2. Information on SEQ ID NO: 5:

(i) sequence characteristics:

(A) Length: 113 base pairs (B) Type: Nucleic acid (C) Fiber: Double-stranded 40 (D) Topology: Linear (ii) Molecule type: DNA (ix) Characteristics:

(A) Name / Key: CDS (B) Location: 1-1113 (xi) Sequence Description: SEQ ID NO: 5

ACT TTG TCC CCG GTC TTC GTG Thr Leu Ser Pro With Phe Va 1 1 5 GTG GTG ACT ATC GTA CTG GCC Va 1 With Thr I le With Leu A1 a 15 20 CAC CAA CGG AGC GGC AGT TAC Hi s Gin Arg Ser Gly Ser Ty r

GCG GTG TTA CTG ACC CTG GGC A1 a Va 1 Leu Leu Thr Leu Gly 10 TTA ATG TAC GTC TTC AGG GAG Leu Me t Ty r With Phe Arg Glu 25 CAT GTT BRANCH 113 Hi s Va 1

35 (2) Information on SEQ ID NO: 6:

(i) sequence characteristics:

(A) Length: 37 amino acids Type 55 (B): Amino acid

Thr Leu Ser Pro Val Phe Val Alá 15 (D) Topology: Linear (ii) Molecule Type: Protein (xi) Sequence Description: SEQ ID NO: 6

Val Leu Leu Thr Leu Gly 10

HU

Va 1 Va 1 Thr I le Va 1 Leu A1 a 15 20 Hi s G1 n 30 Arg Ser Gly Ser Tyr 35

Τ7 176Β 2

Leu Me t Tyr Va 1 Phe Arg Glu

Claims (38)

CLAIMS 1. ICAM-3 and a functional derivative thereof substantially free of natural contaminants, characterized in that the molecular weight of ICAM-3 determined by gel electrophoresis under reducing conditions after immunoprecipitation is M _r = 124,000 and after treatment with N-glycanase M _r = 87,000, and ICAM-3 and its functional derivative bind to LFA-1. (Priority of Amendment: 07/07/1994) 2. A recombinant DNA molecule, characterized in that it encodes ICAM-3 or a functional derivative thereof according to claim 1. (Priority of Amendment: 07/07/1994) Recombinant DNA molecule according to claim 2, characterized in that it is capable of expressing ICAM-3 or a functional derivative thereof according to claim 1 in a cell. (Priority of Amendment: 07/07/1994) ICAM-3 or a functional derivative thereof according to claim 1, characterized in that it is useful as a medicament. (Priority of Amendment: 07/07/1994) A polyclonal or monoclonal antibody or antibody fragment capable of binding ICAM-3 or a functional derivative thereof according to claim 1, characterized in that it is useful as a medicament. (Priority of Amendment: 07/07/1994) 6. A pharmaceutical composition comprising ICAM-3 or a functional derivative thereof according to claim 1 or a polyclonal or monoclonal antibody or antibody fragment capable of binding ICAM-3 or a functional derivative thereof according to claim 1 or a toxin-derived derivative thereof. containing a pharmaceutical carrier or excipient. (Priority of Amendment: 07/07/1994) 7. The pharmaceutical composition of claim 6 further comprising an immunosuppressive agent. (Priority of Amendment: 07/07/1994) Pharmaceutical composition according to claim 6 or 7, characterized in that it is formulated for enteral, parenteral, preferably intramuscular, intravenous or subcutaneous or topical, inhalation or intranasal administration. (Priority of Amendment: 07/07/1994) 9. A 6-8. A pharmaceutical composition according to any one of claims 1 to 3, wherein the functional derivative of ICAM-3 is a peptide according to SEQ ID NOs: 2, 4 or 6 or ICAM-3 domain 1, 1-2. 1-3. 1-4. or domains 1-5. domain. (Priority of Amendment: 07/07/1994) 10 10. A 6-9. Pharmaceutical composition according to any one of claims 1 to 3, characterized in that it is for therapeutic or prophylactic purposes to modify the biological function of a cell mediated by ICAM-3, preferably an inflammatory response, a hematopoietic 15 tumor cell metastases, which require a functional member of the CD18 family to migrate, are capable of modifying the extravascular migration of leukocytes infected with the virus, preferably HIV, or the migration of cells associated with the asthmatic response. 20 (Priority of Amendment: 01/07/1994) 11. The pharmaceutical composition of claim 10, wherein said inflammatory response is a delayed-type hypersensitivity reaction, symptoms of psoriasis, autoimmune disease, organ transplantation, or It is used to treat specific inflammatory responses to tissue rejection. (Priority of Amendment: 07/07/1994) Pharmaceutical composition according to claim 11, characterized in that it is suitable for the treatment of a specific inflammatory response to a solid organ transplantation, preferably a kidney transplant. (Priority of Amendment: 07/07/1994) Pharmaceutical composition according to claim 11, characterized in that it is not solid as an organ transplant 35 organs, preferably for the treatment of a specific inflammatory response to bone marrow transplantation. (Priority of Amendment: 07/07/1994) 14. The pharmaceutical composition according to claim 11, wherein the autoimmune disease is used to treat Rey40 syndrome, autoimmune thyroiditis, EAE, multiple sclerosis, rheumatoid arthritis or erythematous lupus. (Priority of Amendment: 07/07/1994) The pharmaceutical composition of claim 10, Characterized by an inflammatory response to adult respiratory distress syndrome (ARDS), multiple organ injury following blood poisoning, trauma or bleeding, reperfusion injury to myocardial or other tissues, acute glomerulonephritis, reactive arthritis, 50 inflammatory components associated with dermatosis, acute purulent meningitis, or other inflammatory diseases of the central nervous system, such as stroke, heat injury, hemodialysis, leucapheresis, ulcerative colitis, crohn's disease, gonadal ulcer, gonorrhagic for the treatment of a specific inflammatory response. (Priority of Amendment: 07/07/1994) 16. A 6-9. Pharmaceutical composition according to any one of claims 1 to 3, characterized in that you are therapeutic HU217 176Β is used for prophylactic purposes to suppress HIV, preferably HIV-1 infection of leukocytes. (Priority of Amendment: 07/07/1994) 17. A 6-9. Pharmaceutical composition according to any one of claims 1 to 3, characterized in that it is capable of suppressing the growth of a tumor cell expressing ICAM-3 for therapeutic or prophylactic purposes. (Priority of Amendment: 07/07/1994) 18. Pharmaceutical composition according to any one of claims 1 to 3, characterized in that it is capable of suppressing the growth of a tumor cell expressing LFA-1 for therapeutic or prophylactic purposes. (Priority of Amendment: 07/07/1994) 19. 6-18. A pharmaceutical composition according to any one of claims 1 to 6, further comprising an LFA-1 binding antibody or fragment thereof, an LFA-1 non-immunoglobulin antagonist, an ICAM-1 binding antibody or fragment thereof, or an ICAM-2 binding antibody or fragment thereof. . (Priority of Amendment: 07/07/1994) 20. A diagnostic composition comprising ICAM-3 or a functional derivative thereof according to claim 1, which is detectably labeled with a polyclonal or monoclonal antibody or antibody fragment. (Priority of Amendment: 07/07/1994) 21. A method for detecting a cell expressing ICAM-3 comprising: a) incubating the cell or extract thereof in the presence of a nucleic acid molecule hybridizing to ICAM-3 mRNA, and b) detecting a nucleic acid molecule hybridized to a complementary nucleic acid molecule in the cell or cell extract. (Priority: June 11, 1991) 22. A polyclonal or monoclonal antibody or antibody fragment capable of binding to ICAM-3 or a functional derivative thereof according to claim 1, wherein said preparation is a method of diagnosing a tumor cell expressing ICAM-3 in a mammal. (Priority of Amendment: 07/07/1994) 23. A method for diagnosing inflammation in a mammalian fluid sample comprising: a) incubating a sample of the patient with a composition comprising a polyclonal or monoclonal antibody or fragment thereof capable of binding ICAM-3 or a functional derivative thereof according to claim 1, and b) detecting the antibody bound to the fluid, wherein the sample is a tissue sample. (Priority: June 11, 1991) 24. A method of diagnosing the presence of ICAM-3 in a mammalian fluid sample comprising: a) incubating a sample of the patient with a composition comprising a polyclonal or monoclonal antibody or fragment thereof capable of binding ICAM-3 or a functional derivative thereof according to claim 1, and b) detecting the antibody bound to the fluid, wherein the sample is a biological fluid such as blood, serum, plasma, joint fluid, amniotic fluid, spinal fluid or urine. (Priority: June 11, 1991) 25. The functional derivative of ICAM-3 according to claim 1, characterized in that the peptide according to SEQ ID NOs: 2, 4 or 6 or ICAM-3 domain 1, 1-2. 1-3. 1-4. or domains 1-5. domain. (Priority of Amendment: 07/07/1994) 26. A DNA fragment according to claim 2, characterized in that it corresponds to SEQ ID NO: 1, 3 or 5. (Priority of Amendment: 07/07/1994) 27. A method for identifying an agent for modulating ICAM-3 binding to LFA-1, comprising contacting ICAM-3 with LFA-1 in the presence of the agent and measuring the change in ICAM-3 / LFA-1 binding. . (Priority: June 11, 1991) 28. A process for the preparation of ICAM-3 or a functional derivative thereof according to claim 1, wherein the ICAM-3 is isolated from its natural source or recombinantly prepared and purified from its natural impurities. (Priority: June 11, 1991) 29. A method of producing a recombinant DNA molecule encoding ICAM-3 or a functional derivative thereof according to claim 1, wherein the DNA molecule encoding ICAM-3 or a functional derivative thereof is recombinantly assembled with another DNA molecule. (Priority: 6/6/1991) 30. The method of claim 29, wherein the DNA molecule encoding ICAM-3 or a functional derivative thereof is constructed with a DNA molecule that facilitates expression in a cell. (Priority: June 11, 1991) 31. A method of producing a polyclonal or monoclonal antibody or antibody fragment capable of binding ICAM-3 and / or a functional derivative thereof, comprising immunizing a host with ICAM-3 and / or an antigen-specific derivative thereof to ICAM-3 and / or a functional derivative thereof. binding antibodies are recovered from the host and optionally using polyclonal or monoclonal antibodies capable of binding to ICAM-3 and / or a functional derivative thereof using cells that produce the antibodies. (Priority: June 11, 1991) 32. A process for the preparation of a pharmaceutical composition comprising a ICAM-3 or a functional derivative thereof according to claim 28 or a polyclonal or monoclonal antibody or antibody fragment thereof according to claim 31 or a toxin-derived derivative thereof with a pharmaceutical carrier or excipient. stirred. (Priority: June 11, 1991) HU 217 176 Β 33. The method of claim 32, wherein the further component is a pharmaceutical composition comprising an immunosuppressive agent. (Priority: June 11, 1991) 34. The method of claim 32 or 33, wherein the pharmaceutical composition is formulated for enteral, parenteral, preferably intramuscular, intravenous or subcutaneous, or topical, inhalation or intranasal administration. (Priority: June 11, 1991) 35. A 32-34. A process according to any one of claims 1 to 4, wherein the functional derivative of ICAM-3 is a peptide according to SEQ ID NOs: 2, 4 or 6 or ICAM-3 domain 1, 1-2. 1-3. 1-4. or domains 1-5. a. (Priority: June 11, 1991) 36. A method of producing a diagnostic composition, wherein the polyclonal or monoclonal antibody or antibody fragment produced according to claim 31 and detectably labeled. Mixed with 5 carriers or excipients. (Priority: June 11, 1991) 37. The method of claim 28, wherein the functional derivative of ICAM-3 is 2, A peptide according to SEQ ID NO: 4 or SEQ ID NO: 6 10 or ICAM-3 Domain 1, 1-2. 1-3. 1-4. or domains 1-5. . (Priority: June 11, 1991) 38. The method of claim 29, wherein the DNA molecule is SEQ ID NO: 1, 3, or 5.