KR100333120B1 - Intercellular adhesion molecule-3 and its binding ligands - Google Patents

Abstract

The present invention relates to an intercellular adhesion molecule (ICAM-3) involved in the process by which leukocytes recognize and migrate to sites of inflammation and also attach to cell substrates during inflammatory responses. The present invention relates to the molecule, a search method for identifying the molecule, and an antibody capable of binding to the molecule. The present invention also encompasses methods of therapeutic use and diagnostic uses of adhesion molecules and antibodies capable of binding to them.

Description

INTERCELLULAR ADHESION MOLECULE-3 AND ITS BINDING LIGANDS

This application is a continuation of part of US Patent Application Serial No. 07 / 712,879, filed June 11, 1991; and US Patent Application Serial No. 07 / 045,963, filed May 4, 1987, filed 07. / 115,798 (filed November 2, 1987), 07 / 155,943 (filed February 16, 1988), 07 / 189,815 (filed May 3, 1988) and 07 / 250,446 (1988) 07 / 454,294 (filed Dec. 22, 1989) and PCT Application Nos. PCT / US91 / 02942 and PCT / US91 / 02946 (filed April 27, 1991 to the U.S. Repair Office) And the above applications are incorporated herein by reference.

A. Leukocyte adhesion and function

Leukocytes must be able to attach to cell substrates to adequately defend the host against foreign chip particles such as bacteria or viruses, and for a review of these functions, see Eisen, HW's "Microbiology, Third Edition, Harper, Philadelphia, Pennsylvania." Rows 1980, pp. 290-295 and 381-418. White blood cells must be able to attach to endothelial cells so that they can migrate from the circulation to the site of inflammation. In addition, leukocytes must attach to antigen-providing cells so that a normal specific immune response can occur, and finally leukocytes must attach to appropriate target cells to allow lysis of virus infected or tumor cells.

Recently, leukocyte surface molecules involved in mediating this adhesion mediating action have been identified using hybridoma technology. In short, human T-cells (Davignon, D et al. Proc. Natl. Acad. Sci. US 78: 4535-4539 (1981)) and mouse spleen cells (Springer, T et al. Eur. J. Immunol. 9: 301-306 (1979) have been identified, which bind to the surface of white blood cells and inhibit the adhesion related function (Springer, T et al., Fed. Proc. 44: 2660-2663 (1985)). The molecules identified by these antibodies were named Mac-1 and Lymphocyte Function-Related Antigen-1 (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes (Smuninger, T. A. et al., Immunol. Rev. 68: 111-135 (1982)). The two molecules and the third molecule, p150, 95 (having a tissue distribution similar to Mac-1), play a role in cell attachment (Kurger, G et al. Eur. J. Immunol. 15: 1142-1147 (1985)).

The leukocyte molecule includes a related glycoprotein family named "CD-18 family" of glycoproteins (J. Exper, Med. 158: 1785-1803 (1983) by Sanchez-Madrid, F. et al .; Kaiser, GD et al. Eur. J. Immunol 15: 1142-1147 (1985)). This glycoprotein group consists of a heterodimer having one alpha chain and one beta chain. The alpha chains of each of the antigens were different from each other, but the beta chains were found to be highly conserved (J. Exper, Med. 158: 1785-1803 (1983) by Sanchez-Madrid, F et al.). The beta chain of the glycoprotein family (sometimes referred to as CD 18) was found to have a molecular weight of 95 kd, while the alpha chain was found to have a molecular weight of 150 kd to 180 kd (Springer, T., Fed. Proc. 44: 2660-2663 (1985). Although the alpha subunits of the membrane proteins do not share extensive homology with the beta subunits, detailed analysis of the alpha subunits of glycoproteins have shown significant similarities between them. A review of the similarities between the alpha and beta subunits of LFA-1 related glycoproteins is described by Sanchez-Madrid, F. et al., J. Exper.Med. 158: 586-602 (1983); J. Exper.Med. 158: 1785-1803 (1983).

A population of humans has been identified that is unable to express normal amounts of any member of the attached protein family on their white blood cell surface (Anderson, DC et al., Fed, Proc. 44: 2671-2677 (1985); Anderson, DC et al. J. Infect. Dis. 152: 668-689 (1985)). Such people are said to suffer from "leukocyte adherence deficiency disease" (LAD) (Fed. Proc. 44: 2671-2677 (1985) by Anderson, DC; J. Infect, Anderson, DC, et al. Dis, 152: 668-689 (1985)). Characteristics of LAD patients include necrotic soft tissue lesions, impaired pu formation and wound fusion, as well as abnormalities of adhesion-dependent leukocyte function in vitro, and susceptibility to chronic and recurrent bacterial infections. Granulocytes of these LAD patients behave in vitro in the same incomplete manner as normal granulocytes behave in the presence of anti-CD18 monoclonal antibody. That is, they are unable to perform attachment related functions such as aggregation or adhesion to endothelial cells. However, a more important observation is that these patients cannot develop a normal inflammatory response because their granulocytes cannot attach to the cell substrate. The most surprising observation is that granulocytes from these LAD patients cannot reach inflammatory sites, such as skin infections, because they cannot attach to endothelial cells near inflammatory lesions. Such attachment is an essential step of extravascular outflow.

Lymphocytes of these patients showed in vitro defects similar to those of normal subjects, and the CD-18 molecular family of normal subjects was antagonized by antibodies. In addition, these individuals are unable to elicit normal immune responses due to their inability to adhere to the cell substrate (Fed. Proc. 44: 2671-2677 (1985) by Anderson, DC; J. Anderson, DC, et al. Infect. Dis. 152: 668-689 (1985)). These data show that the immune response is alleviated when lymphocytes are unable to attach to the normal type due to the lack of functional adhesion molecules of the CD-18 family.

Thus, in summary, the ability of leukocytes to maintain the health and viability of animals requires their ability to attach to other cells (eg endothelial cells). This attachment has been found to require cell-cell contact involving specific receptor molecules present on the cell surface of leukocytes. These receptors allow leukocytes to attach to other white blood cells, endothelial cells, and other non-vascular cells. The cell surface receptor molecules LFA-1, Mac-1 and p150,95 were found to be highly related to each other. Persons whose leukocytes do not have such cell surface receptor molecules show chronic and recurrent infections, as well as other clinical symptoms, including defective antibody responses.

In addition, because leukocyte adhesion is involved in the process of identifying and rejecting foreign tissues, an understanding of this process is very important in the field of solid organ transplantation such as kidneys, non-solid organ transplantation such as bone marrow, tissue transplantation, allergy and oncology. Has value.

B. HIV infection

HIV infection is the cause of AIDS. Many variants of HIV are disclosed: the two main ones are HIV-1 and HIV-2. HIV-1 is prevalent in North America and Europe, and HIV-2 is prevalent only in Africa. The viruses have similar structures and encode proteins with similar functions. HIV infection is believed to occur by binding of the virus protein ("gp120") to receptor molecules ("CD4") present on the surface of T4 ("T helper") lymphocytes (Schnitman, SM et al. J. Immunol) 141: 4181-4186 (1988), incorporated herein by reference). After binding to the receptor, the virus enters the cell and replicates, killing T cells in the process. Thus, destruction of the T4 community of individuals is a direct result of HIV infection.

Destruction of T cells results in impairment of the infected patient's ability to resist opportunistic infections. People with AIDS often develop cancer, but the relationship between these cancers and HIV infection is uncertain in most cases.

Although simple replication of HIV viruses is fatal to infected cells, such replication is usually only detected in a portion of the T4 cells of an infected person. Recent results suggest that more virusemia occurs than previously assessed, and that the frequency of T cell infections can be as high as 1%.

Several lines of research have identified other mechanisms by which HIV viruses mediate the destruction of the T4 community.

Apart from HIV replication, HIV infected cells can be destroyed through the action of cytotoxic killer cells. Killer cells normally exist in humans and monitor the host and act to destroy any foreign cells they encounter (such as in inappropriate transfusion or organ transplantation). When infected with HIV, T4 cells exhibit gp120 molecules on their cell surface. Killer cells recognize the T4 cells as foreign (rather than the original) cells and thus mediate their destruction.

HIV infection can also result in the destruction of uninfected healthy cells. Infected cells can secrete gp120 protein into the blood system. The free gp120 molecule can bind to the CD4 receptor of healthy, uninfected cells. Such binding gives the cells the appearance of HIV infected cells. Cytotoxic killer cells recognize gp120 bound to uninfected T4 cells and determine that the cells are exogenous and mediate their destruction.

The additional mechanism by which HIV can cause T4 cell death, and the mechanism of particular interest according to the present invention, takes place through the formation of “complexes”. A "complex" is a multinuclear giant cell formed from the fusion of hundreds of T4 cells. Infection with HIV gives the infected cells the ability to fuse with other T cells, ie HIV infected or uninfected healthy cells. The complex cannot function and soon dies. Killing of the corpuscles destroys both HIV-infected and non-HIV-infected T4 cells. This process gives particular attention to the present invention because it causes direct cell-cell contact of T4 cells. The ability of an HIV-infected cell to form a complex indicates that the cell acquires the means to fuse with the healthy cell.

HIV infection, and in particular HIV-1 infection, seems to affect cell surface expression of leukocyte integrins, and cell adhesion responses mediated by these heterodimers (J. Clin. Invest. 79: 188 (Pitt, AJ et al. 1987); Hildard, JEK et al. Science 244: 1075 (1989); Valentin, A. et al. J. immunology 144: 934-937 (1990); Rosette, RD et al. Trans. Assoc. American Physicians 102: 117- 130 (1989), incorporated herein by reference). HIV-1 infection increases the isotopic aggregation of U937 cells and increases the cell surface expression of CD18 and CD11b (J. Clin. Invest. 79: 188 (1987) by Petit, A. J. et al.). HIV-1 infected U937 cells attach to IL-1 stimulated endothelial at a higher frequency than uninfected U937 cells; This behavior can be suppressed by treating cells infected with an anti-CD18 or anti-CD11a monoclonal antibody or by treating the endothelial substrate with an anti-ICAM-1 antibody (Trans. Assoc. American Physicians 102: Lausanne, RD, et al. 117-130 (1989). Monoclonal antibodies against CD18 or CD11a have also been found to be able to inhibit the formation of synapses involving phytohemagglutinin (PHA) -stimulated lymphoblasts and persistently infected CD4-negative T cells (Hildred, JEK) Et al., Science 244: 1075 (1989)). Treatment of only virus-infected cells with an anti-CD18 or anti-CD11a monoclonal antibody has been found to have little effect on the formation of synapses, suggesting that the antibodies in principle protect the non-infected target cells from being infected. (Science 244: 1075 (1989) by Hildred, JEK et al .; J. Immunology 144: 934-937 (1990) by Valentin, A. et al.). Valentin et al. (Valentin, A. et al. J. Immunology 144: 934-937 (1990) recently reported that a monoclonal antibody specific for CD18 was formed when co-culture of continuous T cell lines with HIV-1 infected U937 cells. The observations were confirmed by demonstrating that they inhibited.

The mechanism by which monoclonal antibodies specific for CD18 or CD11a protects susceptible cells from fusion with HIV infected cells is unknown, and although not essential to the understanding of the present invention, studies using radiolabeled gp120 contain CD18. It suggests that the heterodimer does not provide a binding site for the virus (Valentin, A. et al. J. Immunology 144: 934-937 (1990)). Thus, HIV infection requires cell-cell interactions, virus-cell interactions that mimic such cell-cell interactions, or both. Cell-cell interactions can result in the delivery of cell-free viruses or the delivery of virus-infected cells through the endothelial barrier. Virus-cell interactions that mimic cell-cell interactions can allow free viruses to attach to or infect healthy cells, or to facilitate or enable both.

The present invention thus derives in part from the observation that HIV infection, and in particular HIV-1 infection, results in increased expression of CD11a / CD18 heterodimer and its binding ligand. This increased expression is important in that it increases the ability of HIV-infected T cells to attach or aggregate to one another (ie, to “isoform aggregate”). Since such isotopic aggregation is observed not to occur between inactive normal leukocytes, the findings indicate that expression of the CD11 / CD18 receptor, ligands such as ICAM-1, or both are required for this aggregation. LFA-1 must bind to ICAM-1 so that isotopic aggregation occurs. As disclosed herein, ICAM-3 is the only member of the ICAM molecular family that is expressed at high levels on resting T-cells. Only anti-ICAM-3 antibodies can block T-cells from attaching to LFA-1 if T-cells are not activated. Therefore, anti-ICAM-3 antibodies can be used to suppress aggregation of T-cells.

In addition, the attachment of infected T-cells to allow HIV-1 to be transferred from one infected cell to healthy cells using an anti-ICAM-3 antibody, and also to infect healthy cells with or facilitate the infection with free viruses. You can interrupt the process.

C. Migration of HIV-infected cells

The migration and spread of leukocytes is important to protect individuals from infection consequences. However, these processes also play a role in the migration and propagation of virus-infected leukocytes. Of particular interest is the migration and transmission of leukocytes infected with HIV. Such migration of cells leads to the formation of extravascular lesions and can cause tumors and other abnormalities.

Histological examination of affected organs reveals lesions of extravascular mononuclear cell infiltrates. Attempts to identify virus-infected cells in such infiltrates in the central nervous system have indicated the presence of HIV-1 infected cells. These studies have shown that HIV-1 viruses are primarily present in monocytes and macrophages, and other cells of this line (F Johnson et al., F Johnson, J. 2: 2970 (1988); J. Amer et al., MH Staller et al.). Med.Assn. 256: 2360 (1986); S. Gartner et al. Science 233: 215 (1986)).

Mechanisms that stimulate the formation of extravascular infiltrates of HIV-1 infected monocytes have not been well established. This mechanism may involve the transport of acellular viruses or the transport of viruses through the intracellular endothelial barrier of infected mononuclear cells.

Because infection with HIV-1 stimulates the adhesion of leukocytes to vascular endothelial cells and the cell surface expression of molecules that facilitate the transfer of leukocytes from blood to extravascular tissue sites (CW Smith et al. J. Clin. Invest. 82: 1746 (1988), incorporated herein by reference), it has been proposed to prevent the spread of HIV infected cells using antibodies that inhibit cell migration (WO90 / 13316).

D. Asthma: clinical characteristics

Asthma is a heterogeneous group of diseases. It is characterized by hypersensitivity to irritation of tracheal bronchus (K, A. B., Allergy and Inflammation, Academic Press, New York (1987)), incorporated herein by reference. Clinically, asthma is confirmed by extensive obstruction of bronchial bronchus, muddy secretions, and seizures of difficulty breathing, coughing and wheezing. The relative role of each of these conditions is unknown, but the overall result is an increase in airway resistance, overexpansion of the lung and thoracic cavity, and an abnormal distribution of aeration and lung blood flow. The disease is identified in the transient period of acute signs between the asymptomatic periods. Acute transient signs can lead to hypoxia and can be fatal. About 3% of the world's population suffers from asthma.

Two types of asthma are disclosed: allergic asthma and specific constitutional asthma. Allergic asthma is usually associated with hereditary allergic diseases such as rhinitis, urticaria, eczema and the like. The condition is characterized by a positive sheath-and-flare response to intradermal injection of airborne antigens (such as pollen, environmental contaminants or occupational contaminants), and an increase in serum levels of IgE. The development of allergic asthma seems to be causally related to the presence of IgE antibodies in many patients. Asthmatic patients who do not exhibit the characteristics described above are considered specific constitutional asthma.

Allergic asthma is believed to be regulated by T and B lymphocytes and rely on an IgE response activated by interaction of antigens in air with mast cell-bound pre-formed IgE molecules. Antigen contact must occur at a concentration sufficient to induce IgE production for an extended time to sensitize the individual. Once sensitized, asthmatic patients may show signs in response to extremely low levels of antigen.

Asthma signs can be exacerbated by the presence and amount of trigger antigens, environmental factors, occupational factors, physical labor and emotional stress.

Asthma may include methylxanthine (e.g. theophylline), beta-adrenergic agonists (e.g. catecholamines, resorcinol, salininin and epidrine), glucocorticoids (e.g. hydrocortisone), degranulation inhibitors of mast cells (i.e. , Chromones such as sodium chromoline) and anticholinergic agents (eg atropine).

Asthma is believed to be associated with the influx of eosinophils into the lung tissue (“eosinophilia”) (J. Allergy Clin. Immunol. 77: 527-537 (1986) by Prigas, E, incorporated herein by reference) ).

The immunological basis of asthma has been revealed from bronchoalveolar lavage studies (Godard, P. et al. J. Allergy Clin. Immunol. 70:88 (1982)), and studies of respiratory lotus root tissues stripped from the epithelium (Flavorhan, NA). J. Appl. Physiol. 58: 834 (1985); Baans, PJ et al. Br. J. Pharmacol. 86: 685 (1985)). Although these studies did not elucidate the mechanisms underlying the immunology of asthma, these studies developed a generally accepted hypothesis related to the immunological etiology of the disease (J. Allergy Clin, Frigas, E. et al. Immunol. 77: 527-537 (1986)).

A clear indication of the pathology of asthma is the massive infiltration of pulmonary parenchyma by eosinophils and the destruction of mucous cilia.

The “eosinophil hypothesis” suggests that eosinophils are attracted to the bronchus to neutralize the harmful mediators released by mast cells in the lung. According to this hypothesis, eosinophils are degranulated so that they are attracted to the bronchus to release cytotoxic molecules. Upon degranulation, eosinophils release enzymes such as histaminase, arylsulfatase and phospholipase D, which neutralize the harmful mediators of mast cells. However, these molecules also promote the destruction of the mucous ciliary body, hindering the purification of bronchial secretions and contributing to lung damage, a hallmark of asthma.

Since asthma is associated with the migration of cells, it has been proposed to use antibodies that inhibit this migration to mitigate the effects of allergens in patients.

The present invention relates to the discovery of a novel intercellular adhesion molecule called ICAM-3, which is involved in the process by which a white blood cell population recognizes and adheres to a cell substrate. ICAM-3 mediates cellular interactions with other lymphocytes, macrophages and neutrophils at sites of inflammation and immune responses.

The present invention relates to a method of using ICAM-3 alone or in combination with ICAM-1, ICAM-2 or both to inhibit intercellular adhesion of granulocytes, lymphocytes or macrophage lineage cells. The use of such molecules provides methods for treating specific and nonspecific inflammation.

The present invention also provides a method of treatment that inhibits infection with HIV, in particular HIV-1, by administering ICAM-3 alone, or in combination with ICAM-1, ICAM-2, or both, to a person exposed to or infected with HIV; It is about prevention method. The present invention therefore provides a method of treating diseases such as AIDS (Acquired Immune Deficiency Syndrome) caused by HIV viruses.

The present invention also relates to a therapeutic method that suppresses migration of HIV-infected cells from the circulation using ICAM-3 alone or in combination with ICAM-1, ICAM-2 or both. The present invention therefore provides a method of treating a disease such as AIDS (Acquired Immune Deficiency Syndrome) caused by HIV-1 virus.

The present invention also relates to a method of treatment of suppressing T cell death and coform formation in HIV infected persons using ICAM-3 alone or in combination with ICAM-1, ICAM-2 or both. The present invention therefore provides a method of treating a disease such as AIDS (Acquired Immune Deficiency Syndrome) caused by HIV-1 virus.

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

The present invention further relates to molecules capable of binding to ICAM-3 (hereinafter referred to as anti-ICAM-3). Binding of anti-ICAM-3 molecules to ICAM-3 is to modulate the biological actions associated with ICAM-3. The binding molecule of the invention may be an antibody, peptide, or carbohydrate capable of binding to ICAM-3. Such binding molecules are used to modulate the biological function of ICAM-3.

The invention also relates to methods of using ICAM-3 alone or in combination with ICAM-1, ICAM-2 or both to inhibit intercellular adhesion of granulocytes, lymphocytes or macrophage lineage cells. The use of such molecules provides methods for treating specific and nonspecific inflammation.

The invention also provides a treatment for suppressing infection of HIV, in particular leukocytes, by HIV-1, by administering ICAM-3 alone, or in combination with ICAM-1, ICAM-2, or both, to a person exposed to or infected with HIV. It relates to a method and a prevention method. The present invention therefore provides a method of treating diseases such as AIDS (Acquired Immune Deficiency Syndrome) caused by HIV viruses.

The invention also relates to a method of treatment that inhibits migration of HIV-1 infected cells from the circulation using anti-ICAM-3 agents alone or in combination with ICAM-1, ICAM-2 or both. The present invention therefore provides a method of treating a disease such as AIDS (Acquired Immune Deficiency Syndrome) caused by HIV-1 virus.

1 Attachment of Cell Lines to Purified LFA-1

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

2 Flow cytometry analysis of cell ICAM-1, ICAM-2 and ICAM-3 expression

(A) Lymphoblast cell lines were treated with MAb RR1 / 1 (anti-ICAM-1, MAb CB-IC2 / 1 (anti-ICAM-2), MAb CBR-IC3 / 1 (anti-ICAM-3) or non-binding vs. Labeled with saturation of crude MAb X63 (thin line) and labeled with FITC-anti-mouse immunoglobulin (B) Human resting leukocytes and pHA-activated lymphocytes for 3 days were analyzed as described above for ICAM expression. .

Immune sedimentation response of ICAM-3

(A) ¹²⁵ I-labeled cell lysates were immunoprecipitated with non-binding control X63 or MAb CB-IC3 / 1 (anti-ICAM-3). (B) Non-binding control MAb X63, MAb W6 / 32 (anti-HLA-A, B, C), MAb, with or without treatment with ¹²⁵ I-labeled SKW3 cell lysates Immunoprecipitated with RR1 / 1 (anti-ICAM-1), MAb CBR-IC2 / 1 (anti-ICAM-2) or MAb CB-IC3 / 1 (anti-ICAM-3).

4 Immune sedimentation response of ICAM-3 from different sources

ICAM-3 was immunoprecipitated from I ¹²⁵ -labeled lymphocytes and neutrophil lysates using mAb-bound sepharose. Immunoprecipitated ICAM-3 was isolated using polyacrylamide gel electrophoresis. Different mAbs were used to immunoprecipitate other cell surface molecules. Human Ig Sepharose (Control Lane), CBR-IC2 / 1 (ICAM-2), CBR-IC3 / 1 (ICAM-3), TS2 / 4 (LFA-1), LM2 / 1 (MAC-1).

5 Effect of PMA on SKW3 binding to ICAM-1 and ICAM-3

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

Figure 6 Antibody Block of PMA Stimulated SKW3 Cell Adhesion

Several antibodies were tested as previously described for the ability to block PMA stimulated SKW3 cells from binding to purified ICAM-1 or ICAM-3 (Nature 341: 619 (1989) by Dustin et al .; Cell 51, Marlin et al. 813-819 (1987). One representative experiment is shown and error bars represent one SD.

Binding of COS Cell Transformants to Purified ICAMs

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

8 Temperature dependence of PMA stimulated SKW3 binding to ICAM

The ability of PMA stimulated SKW3 cells to bind purified ICAM-1 and ICAM-3 was tested at 4, 16, 22 and 37 ° C. as previously disclosed (cells 51: 813-819 (1987), such as dried). Analyzes were performed with or without LFA-1 mAb (TS1 / 22). One representative experiment is shown and error bars represent one SD.

Figure 9 Antibody Block of PHA Stimulated T Cell Division

Various antibodies were tested as previously disclosed for their ability to block PHA stimulated T cell division (J. Immunol. 131: 611-616 (1983)).

Figure 10 Influence of anti-ICAM antibodies on mixed lymphocyte response

Several antibodies were tested as previously disclosed for their ability to block mixed lymphocyte responses (J. Immunol. 131: 611-616 (1983)).

11 Gas Chromatograph of Peptide Fragments of ICAM-3

ICAM-3 was purified and digested with Lys-C. Peptide fragments were separated using high performance liquid chromatography.

Schematic representation of clone 11.2

Schematic representation of clone 11.2. The positions of the NK-10 and NK-17 peptides, the various DNA sequences thus obtained, and the transmembrane regions are shown.

Figure 13 Sequence comparison of sequences primed with human ICAM-1 and NK-10

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

Fig. 14 Sequence comparison of human ICAM-1 with sequences primed with RM13

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

15 Sequence comparison of human ICAM-2 with sequences primed with RM13

The GAP program was used to identify sequence homology of human ICAM-2 with sequences primed with RM13.

Fig. 16 Sequence primed with T7 and sequence of human ICAM-1

The GAP program was used to identify sequence homology of human ICAM-1 with sequences primed with T7.

17 Sequence comparison of human ICAM-2 with sequences primed with T7

The GAP program was used to identify sequence homology of human ICAM-2 with sequences primed with T7.

Figure 18 Partial Sequence of ICAM-3

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

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

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

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

The invention also relates to the use of anti-ICAM-3 agents alone or in combination with anti-ICAM-1, anti-ICAM-2 or both in the treatment of asthma.

Summary of the Invention

The present invention is based on the discovery of a novel cell adhesion molecule named Intercellular Adhesion Molecule-3 (ICAM-3). The present invention is further capable of binding to functional derivatives of ICAM-3, anti-ICAM-3 antibodies, anti-ICAM-3 antibodies to which the antibody fragments have been applied to the human body, and ICAM-3 and inhibiting their biological function. It is about other molecules that can be.

The invention also encompasses diagnostic and therapeutic uses for all of these molecules.

Specifically, the present invention includes ICAM-3, or functional derivatives of ICAM-3 that are substantially free of natural contaminants.

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

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

The present invention also provides hybridoma cells capable of producing such monoclonal antibodies.

The present invention provides a method for producing a target hybridoma cell for producing an antibody capable of binding to ICAM-3 or a functional derivative thereof, the method comprising the following steps:

(a) substantially pure ICAM-3, a cell expressing ICAM-3, a membrane of cells expressing ICAM-3, ICAM-3 bound to a carrier, a peptide fragment of ICAM-3, or ICAM- bound to a carrier Immunizing the animal with an immunogen selected from the group consisting of peptide fragments of 3,

(b) fusing the splenocytes isolated from said immunized animal with a myeloma cell line,

(c) allowing the fused spleen and myeloma cells to form antibody secreting hybridoma cells, and

(d) searching for hybridoma cells capable of producing anti-ICAM-3 antibodies among the hybridoma cells.

The invention also provides a method of modulating the ICAM-3 mediated biological action of a cell, the method comprising providing an effective amount of an ICAM-3 modulating agent to a patient in need of such action. 3 modulating agents include antibodies capable of binding to ICAM-3; A fragment of the antibody capable of binding ICAM-3; ICAM-3; Functional derivatives of ICAM-3; And non-immunoglobulin antagonists of ICAM-3 that are different from members of the ICAM-1, ICAM-2 or CD-18 molecular family.

The present invention also provides a method of treating specific inflammation in humans and other mammals, the method comprising providing an anti-inflammatory agent in an amount sufficient to suppress inflammation in a patient in need thereof. When the anti-inflammatory agent is an antibody capable of binding to ICAM-3; A fragment of the antibody capable of binding ICAM-3; Substantially pure ICAM-3; A functional derivative of ICAM-3, or a non-immunoglobulin antagonist of ICAM-3, wherein the inflammation is a solid organ transplant (e.g. kidney), a non-solid organ transplant (e.g. bone marrow) or tissue transplant Is the result.

The invention also provides a method of treating nonspecific inflammation in humans and other mammals, the method comprising providing an anti-inflammatory agent in an amount sufficient to suppress inflammation in a patient in need of such treatment The anti-inflammatory agent may be an antibody capable of binding to ICAM-3; A fragment of the antibody capable of binding ICAM-3; Substantially pure ICAM-3; Functional derivatives of ICAM-3, or non-immunoglobulin antagonists of ICAM-3.

The present invention further provides the above method of treating inflammation, wherein the inflammation is adult respiratory distress syndrome; Multiple organ injury syndrome dependent on sepsis; Multiple organ injury syndrome dependent on sepsis, bleeding or trauma; Reperfusion injury of myocardial and other tissues; Acute glomerulonephritis; Reactive arthritis; Skin diseases with acute inflammatory sites; Acute purulent meningitis or other central nervous system inflammatory disease (eg, seizures); Thermal damage; Hemodialysis; Leukocyte clearing blood transfusion; Ulcerative colitis; Crohn 's disease; Necrotic small intestine colitis; Granulocyte transfusion related syndrome; And cytokine-induced toxicity.

The invention also includes a method of suppressing the metastasis of hematopoietic tumor cells, ie, cells that use a member of CD-18 (especially LFA-1) for migration, which method can be used in patients in need of suppression of such metastasis. Providing an amount of the agent sufficient to suppress metastasis, wherein the agent comprises an antibody capable of binding to ICAM-3; Toxin-derived derivatives of this antibody; A fragment of the antibody capable of binding ICAM-3; Toxin-derivatives of such fragments; Substantially pure ICAM-3; Toxin-derived ICAM-3; Functional derivatives of ICAM-3; Toxin-derivatives of these functional derivatives of ICAM-3; Or a non-immunoglobulin antagonist of ICAM-3 that is different from a member of the CD-18 molecular family.

The invention also includes a method of suppressing the proliferation of ICAM-3 expressing tumor cells, the method comprising providing a patient in need of such proliferation with an amount sufficient to suppress said proliferation. When the agent is an antibody capable of binding to ICAM-3; Toxin-derived derivatives of this antibody; A fragment of the antibody capable of binding ICAM-3; Toxin-derivatives of such fragments; Toxin-derived members of the CD-18 molecular family; Or toxin-derived functional derivatives of members of the CD-18 molecular family.

The present invention also provides a method for detecting the presence of cells expressing ICAM-3, the method comprising the following steps:

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

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

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

(a) incubating the sample with an antibody or antibody fragment capable of binding ICAM-3; And

(b) detecting whether the antibody is bound to the sample.

The present invention also relates to (a) an antibody capable of binding to ICAM-3; A fragment of the antibody capable of binding ICAM-3; Substantially pure ICAM-3; Functional derivatives of ICAM-3; Or an agent selected from the group consisting of non-immunoglobulin antagonists of ICAM-3 different from a member of the CD-18 molecular family, or (b) a pharmaceutical composition comprising an immunosuppressant.

Description of Preferred Embodiments

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

I. LFA-1

The leukocyte adhesion molecule LFA-1 mediates the interaction of lymphocytes, monocytes, natural killer cells and granulocytes with other cells in immune and inflammation (Springer, TA et al. Ann. Rev. Immunol. 5: 223-252 (1987). )).

LFA-1 is a receptor for ICAM-1, ICAM-2 and newly identified ICAM-3 (disclosed herein). These surface molecules are structurally expressed in some tissues and induced in inflammation in other tissues (cells 51: 813-819 (1987), such as dried and SD; J. Immunol. 137: 245-254 (Dustin, ML, etc.). 1986; Immunol, Dustin, ML, et al. Today 9: 213-215 (1988); US Patent Application Serial No. 07 / 019,440 (filed February 26, 1987) and US Patent Application Serial No. 07 / 250,446 ( Application, Sep. 28, 1988), which is incorporated herein by reference.)

LFA-1 functions in antigen-specific and antigen-independent interactions with T cell toxicity, T helpers, natural killer, granulocytes, and other cell types of monocytes (Springer, TA et al. Ann. Rev. Immunol. 5: 223- 252 (1987), Kishimoto, TK et al., Adv. Immunol. (1988, published).

II. ICAM-1

ICAM-1 is a single-chain glycoprotein that differs in mass from 76-114KD in different cell types and is a member of the Ig superfamily with five C-like domains (Dustin, ML et al. Immunol. Today 9: 213 -215 (1988); Stoneton, DE et al. Cell 52: 925-933 (1988); and Simmons, D. et al. Nature 331: 624-627 (1988)). ICAM-1 can be induced in large quantities with cytokines including IFN-g, TNF and IL-1 on a wide range of cell types (Dinstin, 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 (J. Immunol. 137: 245-254 (1986) by Dustin, ML; J by Dustin, ML et al. Cell Biol. 107: 321-331 (1988); Dustin, ML et al. J. Exp. Med. 167: 1323-1340 (1988). Adhesion is blocked by pretreatment of lymphocytes with LFA-1 MAb or by pretreatment of other cells with ICAM-1 MAb (J. Immunol. 137: 245-254 (1986) by Dustin, ML; Dustin , ML et al., J. Cell. Biol. 107: 321-331 (1988); Dustin, ML et al. J. Exp. Med. 167: 1323-1340 (1988)). The same results using purified ICAM-1 in artificial membranes or on Petri dishes demonstrate that LFA-1 and ICAM-1 are receptors for each other (cells 51: 813-819 (1987); Bar, MW et al. Nature 331: 86-88 (1988)). For clarity, they are referred to herein as ?? receptors ?? and ?? ligand ??, respectively. Further description of ICAM-1 is described in US patent application Ser. No. 07 / 045,963; No. 07 / 115,798; No. 07 / 155,943; No. 07 / 189,815 or 07 / 250,446, which applications are incorporated herein by reference.

III. ICAM-2

Other LFA-1 ligands have been hypothesized to distinguish from ICAM-1 (J. Immunol. 137: 1270-1274 (1986) by Rhodeline, R. et al .; Eur. J. Immunol. 18: 637 by Makgoba, MW et al. -640 (1988); Dustin, ML et al. J. Cell. Biol. 107: 321-331 (1988)). The identified second LFA-1 ligand is named ICAM-2.

ICAM-2 differs from ICAM-1 in cell distribution and in lack of cytokine induction. ICAM-2 is an integrated membrane protein with two Ig-like domains, while ICAM-1 has five Ig-like domains (Cell 52: 925-933 (1988); Simmons, D. et al. Nature 331: 624-627 (1988). Surprisingly, ICAM-2 is much closer (34% identity) to the two N-terminal domains present at the most N-terminus of ICAM-1 than ICAM-1 or ICAM-2 is to other members of the Ig phase. There is a relationship, which demonstrates that ICAM-2 is a subfamily of Ig-like ligands that bind to the same integrin receptor. Further description of ICMA-2 is provided in US Patent Application Serial No. 07 / 454,294, which is incorporated herein by reference.

IV. ICAM-3

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

The development of MAbs for ICAM-2 enabled the analysis of several conventional LFA-1 dependent, ICAM-1 independent phenomena, suggesting the presence of a third ligand for LFA-1. Binding of purified LFA-1 to various cell types, such as epithelial and endothelial cells, can be completely blocked by a combination of ICAM-1 and ICAM-2 MAbs, whereas ICAM-1, ICAM- of attachment to LFA-1 2-independent pathways were present in many lymphocyte cell lines, including the T cell lymphoma cell line, SKW3 (FIG. 1).

To elucidate this adhesion pathway, MAbs were formed against SKW3 and searched for their ability to inhibit the cell line binding to purified LFA-1 in combination with anti-ICAM-1 and anti-ICAM-2 MAbs. One MAb, CBR-IC3 / 1, was selected, which completely inhibited this novel adhesion pathway (FIG. 1). The adhesion of SKW3 to purified LFA-1 was only slightly inhibited by the combination of blocking anti-ICAM-1 and anti-ICAM-2 MAbs. When only the anti-ICAM-3 MAb, CB-IC3 / 1, was added, it greatly inhibited adhesion, which inhibition was complete when combined with blocking anti-ICAM-2 MAb. Thus, the attachment of SKW3 to purified LFA-1 was predominantly mediated by ICAM-3 and also partially mediated by ICAM-2. When attaching to LFA-1, each of the four cell lines used three ICAMs to different degrees, evidenced by different types of MAb inhibition. Attachment of the B lymphocyte cell line, JY, occurred primarily through the ICAM-1 pathway and slightly through the ICAM-2 and ICAM-3 pathways. Another B lymphocyte cell line, SLA, used both ICAM-1 and ICAM-3 because adhesion was not inhibited by either ICAM-1 or ICAM-3 MAbs, but almost completely by both MAbs. to be. Jurkat, a thymoma cell line, used both the ICAM-2 and ICAM-3 attachment pathways and slightly the ICAM-1 pathway. The adhesion of both resting T lymphocytes and lymphocytes activated with phytohemagglutinin (PHA) was also examined (FIG. 1B). Resting lymphocytes are known to bind strongly to previously purified LFA-1 (Nature 341: 619-624 (1989) by Dustin et al.), And this binding was found to be primarily ICAM-3-dependent. When activated with PHA, ICAM-1 expression was induced and attachment to LFA-1 occurred mainly through ICAM-1 and in part via ICAM-3. Although masked by the presence of ICAM-1 and ICAM-3, ICAM-2 attachment components could be detected in both resting and active T cells. There is a significant excess in the use of ICAMs, as MAbs for two or more ICAMs are required to achieve substantial inhibition for each cell. A notable exception to this is the attachment of resting lymphocytes to LFA-1, which was primarily mediated by ICAM-3. In all cases, the combination of all three anti-ICAM-MAbs prevented binding to LFA-1 at a level comparable to that seen in the presence of anti-LFA-1 MAbs.

The relative affinity of the three ICAMs for LFA-1 can be examined by comparing the action of binding LFA-1 measured above and cell surface expression measured by immunofluorescence flow cytometry (FIG. 2). Comparison of the surface expression of ICAMs with their action on LFA-1 adhesion showed that ICAM-1 had the highest affinity for LFA-1, while ICAM-2 and ICAM-3 had similar but low affinity. . For example, Jurkat cells expressed similar levels of ICAM-2 and ICAM-3 and acted on binding to LFA-1, respectively. When ICAM-2 expression was expressed more than the expression of ICAM-3 as on JY cells, the ICAM-2 pathway of LFA-1 attachment was superior to ICAM-3. In contrast, SLA and SKW3 expressed 3-5 times more ICAM-3 than ICAM-2, and as expected the ICAM-3 attachment pathway was superior to the ICAM-2 pathway. It was well expressed in the presence of ICAM-3 on resting T lymphocytes, and in the absence of ICAM-1, attachment to LFA-1 was primarily mediated through ICAM-3. When ICAM-1 is well expressed, as in the case of SLA, JY and PHA-activated T cells, ICAM-1 was the main adhesion pathway.

The distribution type of ICAM-3 differed in many ways from that of ICAM-1 and ICAM-2. Unlike ICAM-1 and ICAM-2, ICAM-3 was not expressed on resting or stimulated endothelial (data not shown). This is consistent with the finding that LFA-1-dependent binding of cells to resting and stimulated endothelial cells is an ICAM-1 and ICAM-2 dependent phenomenon. ICAM-3 is highly expressed in the hematopoietic lineage, ie lymphocytes and monocyte cell lines with some exceptions. However, in all cases, expression of ICAM-3 in cell lines was comparable to LFA-1-dependent, ICAM-1, and ICAM-2 independent adhesion pathways. Cell lines that bind LFA-1 only through ICAM-1 and ICAM-2 (HUVEC, Raji) did not show ICAM-3 expression, whereas cell lines showing weak binding through the third attachment pathway (JY, U937, Sup T) had correspondingly low ICAM-3 surface expression (data not shown).

ICAM-3 was significantly different from ICAM-1 and ICAM-2 in its expression on white blood cells (Figure 2B). ICAM-3 was expressed at high levels on resting lymphocytes, monocytes and neutrophils, while ICAM-1 and ICAM-2 were much weaker or not expressed. In comparison, ICAM-1 and ICAM-2 were weakly expressed on monocytes and only ICAM-2 was present on resting lymphocytes. Neither ICAM-1 nor ICAM-2 was expressed in neutrophils. When activating lymphocytes with PHA, ICAM-3 expression increased 2-3 fold, whereas ICAM-1 expression was significantly induced (Dustin et al. J. Immunol. 137: 245-254 (1986)) (2B Degree).

ICAM-3 immunoprecipitates of various ¹²⁵ I labeled cell lines showed a sharp band of 124,000 Mr under reducing conditions and only slightly increased mobility under non-reducing conditions (FIG. 3A). N-glycanase treatment reduced the ICAM-3 band by Mr 87,000, suggesting that ICAM-3 is a highly prized protein like ICAM-1 and ICAM-2 (Figure 3B). The biochemical, expression type and functional properties of ICAM-3 are inducible to previously disclosed adhesion molecules such as human regressive receptor LAM-1 (J. Immunol. 144: 532-540 (1990) by Tether et al.), Inducible Endothelial Adhesion Molecule VACAM-1 (Rice et al. J. Exp. Med. 171: 1369-1374 (1990); Carlos et al. Blood (published) (1990); Osborne et al. 59: 1203-1211 (1989)) , And MAb with similar cell distribution, are distinguished from the VLA family of matrix receptors (Helmer, ME et al., Ann. Rev. Immunol. 8: 365-400 (1990)). Leukocyte typing database IV such as WR, Oxford University Press, Oxford, UK (1990)).

The presence of three LFA-1 ligands suggests characterization for different aspects of LFA-1-dependent leukocyte interaction. ICAM-1 is expressed fundamentally in endothelial and many epithelial cell types, which are strongly induced in inflammation and immunity to regulate cell localization and to facilitate recognition of specific antigens (Immunol. Rev. 108: 135-). 161 (1989). Since ICAM-2 is the predominant LFA-1 ligand in resting endothelium, this adhesion pathway may have important consequences for the normal recycling of LFA-1-bearing lymphocytes through tissue endothelium (J. Immunol. 140: 693). -699 (1988); McKay et al. J. Exp. Med. 171: 810-817 (1990); Pallas et al. J. Immunol. 140: 1851-1853 (1988); and Nunoy et al. Hum, Path. 19: 753-759 (1988). Currently, the function of ICAM-3 on neutrophils is not clear, because neutrophil isoform aggregation appears to be primarily Mac-1-dependent despite the presence of LFA-1 (Jander Im et al. 137: 15). -27 (1986); Pattroyo et al. Scand. J. Immunol. 22: 619-631 (1985). The discovery that attachment of resting T lymphocytes to LFA-1 occurs primarily through ICAM-3, with the fact that ICAM-3 is much better expressed on monocytes and resting lymphocytes than other LFA-1 ligands in the initiation of the immune response. Imply an important role. Indeed, T cell attachment to antigen-providing cells requires LFA-1: ICAM interaction (Imm. Rev. 114: 29-44 (1990) by D. Field et al .; Immunol et al. Today 10: 417-422 (1989)), ICAM-3 may play a particular role on resting T lymphocytes, which are themselves poorly expressed in both ICAM-1 and ICAM-2. In fact, a role has been proposed for LFA-1 ligands different from ICAM-1 in allergic and autologous mixed lymphocyte responses (Cell Immunol. 128: 362-369 (1990), Bagnasco et al.), ICAM-3 can be expected to be important for antigen-specific interactions between those two lymphocytes, one of which is not yet activated. ICAM-3 is also dependent on LFA-1 but may be important for lysis of certain targets by T cells but not ICAM-1 (Eur. J. Immunol 18: 637-640 (1988) by Makgoba et al.).

The presence of multiple ICAMs has important implications in clinical treatment with MAbs for ICAM or ICAM analogs. ICAM-1 MAb is used for the persistence of allografts in the kidneys (J. Immunol. 144: 4604-4612 (1990), Kosimi et al., Transplant. Proc. 23: 533-534 (1991), Flavin et al.). It is effective in vivo. ICAM-3 MAb inhibits a number of distinct and redundant immune responses in vivo because it inhibits LFA-1-dependent adhesion interactions of different subsets of cell types.

V. cDNA Cloning of ICAM-3

One of a variety of methods can be used to clone the ICAM-3 gene. One such method involves analyzing a shuttle vector library of cDNA inserts (derived from ICAM-3 expressing cells) for the presence of an insert comprising the ICAM-3 gene. Such assays can be performed by transfecting cells with a vector and analyzing ICAM-3 expression.

ICAM-3 cDNA was modified using the method of Arupo and Seed (Proc. Natl. Acad. Sci. US 84: 3365-3369 (1987)) of Seed, B. et al. It is desirable to identify. In this method, cDNA libraries are prepared from cells expressing ICAM-3 (eg, SLA, Jurkat, or SKW3 lymphoblast cell lines). cDNA libraries are preferably prepared from T-cells. This library is used to transfect cells that do not normally express ICAM-3 (eg Cos or HeLa cells). Transfected cells are introduced into a Petri dish previously coated with LFA-1 or anti-ICAM-3 antibody. Transfected cells comprising the ICAM-3 coding sequence and expressing this ligand on their cell surface will attach to LFA-1 or anti-ICAM-3 antibodies on the Petri dish surface. Non-adherent cells were washed away and adherent cells were transferred to Petri dishes and cultured. The recombinant ICAM-3 expression sequence is determined in these transferred cells.

When LFA-1 is used to coat a Petri plate, anti-ICAM-1 and anti-ICAM-2 specific antibodies are added to the Petri dish to prevent adhesion of ICAM-1 or ICAM-2 expressing cells. Thus, the attachment of ICAM-1 ⁺ or ICAM-2 ⁺ transfectants to LFA-1 coated plastics is such as anti-ICAM-1 MAb RR1 / 1 and anti-ICAM-2 MAb CBR-IC2 / 2. It can be inhibited with an antibody. Binding of ICAM-3 transfected cells to LFA-1 coated Petri plates is inhibited by EDTA and anti-LFA-1 MAbs, but not by anti-ICAM-1 or anti-ICAM-2 MAbs. Therefore, ICAM-1 or ICAM-2 expressing cells cannot attach to Petri dishes and therefore are washed mainly with all other non-adherent cells. This will enrich the cells expressing ICAM-3.

Other methods of preparing gene sequences encoding ICAM-3 are well known in the art and one of ordinary skill in the art can typically employ one of these methods to obtain the desired gene.

One such other method of preparing a gene sequence encoding ICAM-3 is to search for cDNA or genomic DNA libraries using oligonucleotide probes. In this method, the ICAM-3 protein is preferably purified using immuno-purification methods known in the art, and the terminal amino acid sequence is determined using one of the methods known in the art. Meanwhile, ICAM-3 peptides enzymatically washed with trypsin or Lys-C are purified and the amino acid sequence of one of the internal fragments is determined.

Once the partial amino acid sequence is determined, oligonucleotide probes are made based on the codon preferences exhibited by the organism, degenerate probes are made on the basis of all possible codon combinations, or codon preferences, known ICAM-1 or ICAM- A probe is prepared using a combination of homology with 2 DNA sequences and codon degeneracy. This probe is then used to search for genomic libraries or cDNA libraries for sequences that hybridize to these probes.

Techniques similar to or similar to those described above include human aldehyde dehydrogenase (Shoe, LC et al. Proc. Natl. Acad. Sci. US 82: 3771-3775 (1985)), fibronectin (Suzuki, S. et al. Eur. Mol. Biol) Organ. J. 4: 2519-2524 (1985)), human estrogen receptor genes (Walter, P. et al., Proc. Natl. Acad. Sci. US 82: 7889-7893 (1985)), and tissue-type plastics. Successful cloning of the gene for minogen activating factor (Nature 301: 214-221 (1983) by Phenica, D. et al.) Was made possible.

In another method of cloning the ICAM-3 gene, an expression library is prepared by cloning genomic DNA, or more preferably cDNA, from cells expressing ICAM-3. The library is then searched for members capable of expressing proteins that bind to anti-ICAM-3 antibodies.

The cloned ICAM-3 gene obtained using any of the methods described above can be operatively linked to an expression vector and introduced into bacterial or eukaryotic cells to produce the ICAM-3 protein. Such manipulation techniques are disclosed in the literature (see above) by Manistes, T and the like and are well known in the art.

Another method of cloning the ICAM-3 gene using PCR makes the assumption that ICAM-3 is homologous to ICAM-1, ICAM-2, or both. Amplify cDNA or mRNA by polymerase chain reaction from cells known to express ICAM-3 proteins such as SKW3 or amygdala using degenerate oligonucleotide primers based on sequences conserved between ICAM-1 and ICAM-2 (J. Exp. Med., 175: 185-190 (1990) by Defugerowls et al.). Clones were sequenced and clones distinguished from ICAM-1 and ICAM-2 were used to obtain full length cDNA clones.

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

VI. Formulations of the Invention: ICAM-3 and Its Functional Derivatives, Agonists and Antagonists

The invention also relates to ICAM-3, its "functional derivatives", its "functional substances" and "antagonists".

A. Functional Derivatives of ICAM-3

A “functional derivative” of ICAM-3 is a compound that has a biological activity (functional and structural) that is almost similar to that of ICAM-3. The term "functional derivative" includes "fragments", "variants" and "chemical derivatives" of the present ICAM-3 molecules.

"Fragment" of ICAM-3 refers to any polypeptide subset of the molecule. Particularly preferred are fragments of ICAM-3 that have ICAM-3 activity and are soluble (ie not bound to the membrane). Soluble fragments may be preferably calculated by deleting the transmembrane site of the parent molecule, deleting hydrophobic residues or substituting hydrophilic amino acid residues. Methods of identifying such residues are well known in the art.

Preference is given to fragments comprising any number of full Ig-like domains, such as domain 1 (D1), D1 and D2, D1-3, D1-4 and D1-5.

A “variant” of ICAM-3 refers to a molecule that is almost similar in structure and function to the entire molecule or fragment thereof.

When one molecule and another molecule have a nearly similar structure or both molecules have similar biological activity, the molecule is said to be "almost similar" to the other molecule. Thus, if two molecules have similar activity, they are considered to be variants as used herein, even if one molecule has a structure not found in the other or the sequence of amino acid residues is not identical. do.

As used herein, when one molecule contains additional chemical moieties that are not normally part of an autogenous molecule, the molecule is said to be a "chemical derivative" of the other molecule. Such sites may also improve solubility, absorption, biological half-life, and the like of the molecule. In addition, this site may reduce the toxicity of the molecule or eliminate or attenuate unwanted side effects of the molecule. Sites that can mediate these effects are described in Remington's "Pharmaceutical Science (1980)".

"Toxin-derived molecules" constitute a particular class of "chemical derivatives". A "toxin-derived" molecule is a molecule covalently attached to a toxin site (eg, an ICAM-3 or anti-ICAM-3 antibody). The process of coupling such sites to molecules is well known in the art.

Coupling these molecules to cells closes the toxin site to the cell, thereby promoting apoptosis. Any suitable toxin site may be used; Preference is given to using toxins such as lysine toxin, cholera toxin, diphtheria toxin, radioisotope toxin or membrane-channel toxin.

Functional derivatives of ICAM-3 having up to about 100 residues can be conveniently prepared by in vitro synthesis. If desired, such fragments may also be modified using methods known in the art by reacting targeted amino acid residues of the purified or crude protein with organic derivatives capable of reacting with selected side or terminal residues. . The covalently derived derivatives obtained can also be used to identify residues important for biological activity.

Many methods can be used to yield and isolate fragments of ICAM-3. Induction with a bifunctional agent can be used to crosslink ICAM-3 to a water-insoluble support matrix. Alternatively reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and US Pat. No. 3,969,287; 3,691,016; 3,691,016; No. 4,195,128; No. 4,247,642; 4,229,537; And the reactive substrates described in US Pat. No. 4,330,440 are used for protein immobilization.

Functional derivatives of ICAM-3 with altered amino acid sequences can also be prepared by mutating the DNA encoding ICAM-3. Such functional derivatives include, for example, deletions, insertions or substitutions of residues in the amino acid sequence of ICAM-3. Any combination of deletions, insertions and substitutions may be used to yield the final construct, provided that the final construct possesses the desired activity. Mutations that will be made in the DNA encoding the functional derivative should not be arranged so that the sequence is outside the translation frame and preferably does not yield complementary sites that can produce secondary mRNA structures (see European Patent Application Publication No. 75,444). ).

At the genetic level, functional derivatives can be prepared by site-directed mutagenesis of the DNA encoding ICAM-3, the DNA encoding the functional derivative can be produced and then the DNA expressed in recombinant cell culture. have.

The site for introducing a mutation in the amino acid sequence is predetermined, but the mutation itself does not need to be predetermined. For example, to best perform mutations at a given site, random mutagenesis, such as linker scanning mutagenesis, is performed at the target codon or target region to yield a large number of derivatives which are then expressed and optimized for the desired combination of activity. Search for It is well known how to sequence mutations at certain sites in known DNA, for example site-directed mutagenesis.

The preparation of functional derivatives of ICAM-3 in accordance with the invention is preferably carried out by site-directed mutagenesis of DNA encoding ICAM-3 or a previously prepared functional derivative of ICAM-3. Such site-specific mutagenesis techniques are described in the art, as illustrated in the literature, Molecular Cloning of Manitosis, T. et al., Laboratory Manual, Cold Spring Harbor, New York (1982), which is incorporated herein by reference. Well known. Site directed mutagenesis makes it possible to prepare ICAM-3 functional derivatives using specific oligonucleotides encoding the DNA sequence of the desired mutation.

Site directed mutagenesis can be used to perform amino acid deletions, insertions or substitutions. The amino acid sequence deletion range is generally about 1 to 30 residues, more preferably 1 to 10 residues and is usually contiguous. Most preferred deletions were performed to yield the soluble form of ICAM-3. Soluble forms of ICAM-3 are most preferably calculated by deleting the transmembrane of ICAM-3 or the hydrophobic residues in ICAM-3.

Amino acid insertions include insertion of single or multiple amino acid residues within an ICAM-3 coding sequence, as well as terminal fusions with polypeptides of almost unlimited length. Intra-sequence insertion (ie, insertion in a complete ICAM-3 molecular sequence) may generally range from about 1 to 10 residues, more preferably 1 to 5 residues. Examples of terminal insertions include fusing heterologous or homologous signal sequences to a host cell at the N-terminus of the molecule for the purpose of promoting secretion of the derivative from the recombinant host.

Functional derivatives of the third group are those in which one or more amino acid residues in ICAM-3 have been removed and other residues inserted in their place. Such substitutions are preferably carried out according to Table 1 below when it is desirable to fine tune the properties of the ICAM-3 molecules.

The essential change in functional or immunological identity is to select substitutions that are less conservative than those shown in Table 1, i.e. (a) the structure of the polypeptide backbone in the substitution region such as the sheet or helix configuration (b) the molecule at the target site. By selecting other residues that are greater in charge or hydrophobicity or in effect of maintaining the volume of (c) the side chain. Generally low conservative substitutions include (a) when glycine proline, or both, are substituted, deleted or inserted by another amino acid; (b) when the hydrophobic moiety is replaced with a hydrophilic moiety; (c) any other residue is substituted with a cysteine residue or vice versa; (d) when the residue with an electronegative charge is substituted with an residue with an electropositive side chain or vice versa; Or (e) a residue having no large side chain is substituted with a residue having such side chain or vice versa.

Most preferred are substitutions that affect the solubility of ICAM-3. This is most preferably done by replacing hydrophobic amino acids with hydrophilic amino acids.

Mutations designed to enhance the affinity of ICAM-3 can also be induced by introducing amino acid residues present at homologous positions in ICAM-1 or ICAM-2. Similarly, such mutant ICAM-3 molecules may be prepared in which N-linked CHO is deleted at homologous positions in ICAM-1 or ICAM-2.

Prior to implementation, it is difficult to accurately predict the effect of certain substitutions, deletions or insertions on the biological activity of ICAM-3. However, those skilled in the art will be able to evaluate the effect by conventional search analysis. For example, derivatives are usually prepared by linker scanning site-directed mutagenesis of DNA encoding native ICAM-3 molecules. This derivative is then expressed in a recombinant host and optionally purified from cell culture, for example by immunoaffinity chromatography.

The activity of the cell lysate or purified derivatives is then searched by a suitable search assay for the desired properties. For example, changes in the immune properties of functional derivatives such as affinity for a given antibody are measured by competitive immunoassay. Appropriate assays determine changes in immunomodulatory activity. Modifications of protein properties such as redox or thermal stability, biological half-life, hydrophobicity, susceptibility to degradation by proteolytic enzymes, or a tendency to aggregate or multimerize with a carrier are analyzed by methods well known to those skilled in the art.

B. Agonists and Antagonists of ICAM-3

The "agonist" of ICAM-3 is a compound that enhances or enhances the ability of ICAM-3 to carry out biological actions. Examples of such agonists are agents that enhance the ability of ICAM-3 to bind cellular receptors.

The "antagonist" of ICAM-3 is a compound that reduces or inhibits the ability of ICAM-3 to carry out biological actions. Examples of such antagonists include ICAM-1 and ICAM-2, functional derivatives of ICAM-1 and ICAM-2, anti-ICAM-3 antibodies, anti-LFA-1 antibodies and the like.

US Patent Application Serial No. 07 / 045,963, which is incorporated by reference herein; No. 07 / 115,798; No. 07 / 155,943; Cell aggregation assays described in 07 / 189,815 or 07 / 250,446 can be used to determine LFA-1 dependent aggregation, and thus can be used to prepare agents that can affect ICAM-3 / LFA-1 aggregation. I can figure it out. In other words, by using such an assay, the agonists and antagonists of ICAM-3 can be identified. Antagonists may also act by damaging the ability of LFA-1 or ICAM-3 to mediate aggregation. In addition, non-immunoglobulin (ie, chemical) formulations may be examined using the assays described above to determine whether this is an agonist or antagonist of ICAM-3 / LFA-1 mediated aggregation. This aggregation assay is usually performed using peripheral T-cells stimulated with PMA. That is, ICAM-3 mediated aggregation is analyzed using the binding of peripheral blood cells to LFA-1.

C. Anti-ICAM-3 antibody

Preferred immunoglobulin antagonists of the invention are antibodies to ICAM-3, such as CBR-IC3 / 1 disclosed herein. Other suitable anti-ICAM-3 antibodies can be obtained by various methods.

Antibodies to ICAM-3 (or functional derivatives of ICAM-3) may be polyclonal or monoclonal antibodies. Antibodies of the present application may also be humanized by procedures known in the art or by PCT Application Nos. PCT / US91 / 02942 and PCT / US91 / 02946, filed with the US Office of Repair on April 22, 1991. .

Anti-ICAM-3 antibodies can also be prepared by introducing ICAM-3 or peptide fragments thereof into a suitable mammal. Immunized animals will produce polyclonal antibodies in response to this exposure. Peptide fragments of ICAM-3 can be used to obtain region specific antibodies that only react with epitope (s) contained within the peptide fragments used to immunize the animal.

Alternatively, anti-ICAM-3 antibodies can also be prepared using ICAM-3, which is originally expressed on the surface of lymphocytes. Introduction of the cells into a suitable animal by methods such as intraperitoneal injection will result in the production of antibodies capable of binding to members of the ICAM-3 or CD-18 molecular family. If desired, such animal serum can be isolated and used as a source of polyclonal antibodies capable of binding these molecules.

Alternatively, anti-ICAM-3 antibodies may be generated by applying the method of Cellden, R. F. (European Patent Application Publication No. 289,034) or Cellden, R. F. et al. (Science 236: 714-718 (1987)). Specifically, cells of suitable animals (eg mice) are transfected with vectors capable of expressing native ICAM-3 molecules or fragments thereof. The production of ICAM-3 in transfected animal cells will induce an immune response in the animal and produce an anti-ICAM-3 antibody by the animal.

However, in order to yield monoclonal antibodies capable of binding ICAM-3, it is desirable to remove splenocytes from immunized animals in one of the ways described above.

Hybridoma cells obtained in the manner described above can be searched by various methods to identify hybridoma cells that secrete antibodies capable of binding to ICAM-3. In a preferred search assay, such molecules are identified by their ability to inhibit aggregation of ICAM-3-form expressing cells, ICAM-1 and ICAM-2-non-expressing cells. Antibodies that can inhibit such aggregation are then further searched to determine whether to inhibit such aggregation by binding to the ICAM-3 or CD-18 molecular family. Any means capable of distinguishing ICAM-3 from the CD-18 molecular family may be used in the screen. Thus, for example, antigens bound by antibodies may be analyzed by immunoprecipitation and polyacrylamide gel electrophoresis. LFA-1 binds to a member of the CD-18 molecular family from an antibody that binds to ICAM-3 by searching for the antibody's ability to bind to cells that express LFA-1 but not ICAM-3 (or vice versa). It is possible to distinguish between antibodies. The ability of an antibody to bind to cells that express LFA-1 but not ICAM-3 may be detected by methods commonly used by those skilled in the art. Such methods include immunoassays (particularly using immunofluorescence), cell aggregation, filter binding studies, antibody precipitation, and the like.

In a preferred method, antibodies are selected for their ability to bind cells that express ICAM-3 but not to cells that do not express ICAM-3.

In addition to the agonists and antagonists of ICAM-3 described above, other agents that may be used in accordance with the invention in the treatment of inflammation, HIV infection, asthma, and the like include anti-idiotypic antibodies and ICAM- anti-ICAM-3 antibodies. Receptor molecules capable of binding to 3, or fragments of such molecules are included.

Anti-idiotypic antibodies of interest in the present invention can bind competitively (or exclude ICAM-3) with ICAM-3. Such antibodies can be obtained, for example, by generating antibodies to anti-ICAM-3 antibodies and then searching for antibodies for the ability to bind to one of the naturally binding ligands of ICAM-3.

Because molecules of the CD-18 group can bind to ICAM-3, such molecules (for example as heterodimers with alpha and beta subunits or as molecules consisting only of alpha or beta subunits or with one or both of the subunits) Administration of a molecule having a fragment of will compete with the cell for binding to ICAM-3 present on the cell.

Anti-aggregated antibodies of the invention can be identified and titrated in a variety of ways. For example, the ability of antibodies to differentially bind to cells that express ICAM-3 (eg, T-cells) and the ability not to bind to cells that do not express ICAM-3 can be measured. Such cell aggregation assays are described in US Patent Application Serial No. 07 / 045,963, which is incorporated herein by reference; No. 07 / 115,798; No. 07 / 155,943; US 07 / 189,815 or 07 / 250,446. Alternatively, the ability of an antibody to bind ICAM-3 or a peptide fragment of ICAM-3 can be measured. As will be readily understood by those skilled in the art, the above assays are provided for the purpose of providing a variety of possible screening assays for identifying and discriminating antibodies capable of binding ICAM-3 relative to members of the CD-18 molecular family, respectively. May be modified or performed in a different order.

D. Preparation of the Inventive Formulations

The formulations of the present invention are polyclonal antibodies capable of inducing or binding to ICAM-3 by natural methods (e.g., to produce animals, plants, fungi, bacteria, etc. to produce non-immunoglobulin antagonists of ICAM-3). By inducing the animal to produce it; By synthetic methods (eg, by synthesizing ICAM-3, functional derivatives of ICAM-3 or protein antagonists (immunoglobulins or non-immunoglobulins) of ICAM-3); By hybridoma technology (for example for the purpose of producing monoclonal antibodies capable of binding to ICAM-3); Or by recombinant techniques (e.g., for the purpose of producing a formulation of the invention in a variety of hosts (i.e. yeast, bacteria, fungi, cultured mammalian cells, etc.) using recombinant plasmids or viral vectors). It may be. The choice of method of use depends on factors such as ease, desired yield, and the like. However, it is not necessary to use only one of the methods, processes or techniques described above to produce specific anti-inflammatory agents; Certain formulations may also be obtained in combination with the foregoing processes, methods and techniques.

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

The formulations of the invention can be used to modulate various biological activities mediated by ICAM-3.

A. Inflammation suppression

One aspect of the invention derives from the ability of ICAM-3 and its functional derivatives to interact with receptors of the CD-18 molecular family, particularly LFA-1. By the ability of ICAM-3 to interact with members of the CD-18 family of glycoproteins, ICAM-3 may be used to suppress (ie, prevent or attenuate) inflammation.

The term "inflammation" as used herein is meant to encompass both the response of a specific defense system and the response of a non-specific defense system.

The term "specific defense system" as used herein refers to components of the immune system that respond to the presence of specific antigens. When inflammation is caused, mediated or related by the response of a specific defense system, it is said to result from the response of the specific defense system. Examples of inflammation resulting from the response of specific defense systems include responses to antigens such as the rubella virus, autoimmune diseases and delayed hypersensitivity reactions mediated by T-cells (e.g. Mantaux ) Observed in subjects tested as "positive" in the test. Rejection of solid graft tissues and organs (eg, kidney and bone marrow transplants) and chronic inflammatory diseases are another example of inflammatory responses of specific defense systems.

As used herein, the response of a "non-specific defense system" refers to a response mediated by leukocytes without immune memory. Such cells include granulocytes and macrophages. As used herein, inflammation is referred to as being caused by, or mediated by, or associated with, a response of a nonspecific defense system.

Inflammation resulting at least in part from the response of a nonspecific defense system includes adult respiratory distress syndrome (ARDS) or multiple organ damage syndrome dependent on sepsis, trauma or bleeding; Reperfusion injury of the myocardium or other tissues; Acute glomerulonephritis; Reactive arthritis; Skin diseases with acute inflammatory sites; Acute purulent meningitis or other central nervous system inflammatory disease (eg, seizures); Thermal damage; Hemodialysis; Leukocyte clearing blood transfusion; Ulcerative colitis; Crohn 's disease; Necrotizing enterocolitis; Granulocyte transfusion related syndrome; And inflammation associated with conditions such as cytokine-induced toxicity.

As discussed above, the binding of ICAM-3 molecules to members of the CD-18 molecular family is critical for cell adhesion. Through the attachment process, lymphocytes can continuously monitor the animal for the presence of foreign antigens. These processes are usually preferred but also cause solid organ transplant rejection (eg, kidney), non-solid organ transplant rejection (eg, bone marrow), tissue transplant rejection, and many autoimmune diseases. Thus, any means capable of attenuating or inhibiting cell adhesion may include solid organ transplants (particularly kidney transplants), non-solid organ transplants (particularly bone marrow transplants), recipients of tissue transplants or autoimmune patients. It is particularly desirable for.

Monoclonal antibodies against members of the CD-18 molecular family include binding to endothelial cells (Hascard, D. et al., J. Immunol. 137: 2901-2906 (1986)), isotopic attachment (Rodlein, R. et al. , J. Exp. Med. 163: 1132-1149 (1986)). Antigen and mitogen-induced proliferation of lymphocytes (Davignon, D. et al., Proc. Natl. Acad. Sci., US 78: 4535-4539 (1981)), antibody formation (Fisher, A. et al., J. Immunol. 136: 3198-3203 (1986)), and cytotoxic T cells (Crensky, AM et al., J. Immunol. 132: 2180-2182 (1984)), macrophages (Strathman, G. et al., J. Immunol. 136: 4328-4333 (1986)), and the effect of all leukocytes, such as the lytic activity of cells involved in the cytotoxic response of antibody-dependent cells (Cool, S. et al., J. Immunol. 133: 2972-2978 (1984)). It inhibits many of the adhesion-dependent functions of leukocytes, including factor function. In all of these functions, the antibody inhibits the ability of white blood cells to attach to suitable cell substrates that inhibit the biological functions associated with binding. This function can be inhibited by anti-ICAM-3 antibodies to the extent that they include ICAM-3 / LFA-1 interactions.

Thus, monoclonal antibodies capable of binding ICAM-3 can be used as anti-inflammatory agents in mammalian patients. Such agents differ from the general anti-inflammatory agents in that they can selectively inhibit adhesion and do not provide other side effects such as the nephrotoxicity found in conventional formulations.

In particular, soluble ICAM-3 can act in the same way as antibodies against members of the CD-18 family of molecules, so it can also be used to suppress inflammation. In addition, functional derivatives and antagonists of ICAM-3 can also be used to suppress inflammation.

One. Inhibitors of delayed type hypersensitivity reactions

ICAM-3 partially mediates the adhesion process required to initiate an inflammatory response, such as a delayed hypersensitivity reaction. Thus, antibodies (particularly monoclonal antibodies) capable of binding to ICAM-3 have therapeutic potential in weakening or excluding these responses.

Alternatively, since ICAM-3 is an antagonist of ICAM-1 / LFA-1 interactions, ICAM-3 (particularly soluble) or functional derivatives thereof can be used to inhibit delayed hypersensitivity reactions.

These potential therapeutic uses may be developed in one of two ways. First, a composition comprising an anti-ICAM-3 monoclonal antibody or a soluble derivative of ICAM-3 may be administered to a patient with a delayed-type hypersensitivity reaction. For example, such compositions may be provided to an individual who has been in contact with an antigen such as ivy poison, oak poison, and the like. In another embodiment, monoclonal antibodies capable of binding ICAM-3 are administered to the patient with the antigen to prevent subsequent inflammatory response. Thus, further administration of an antigen with an anti-ICAM-3 antibody may serve to temporarily tolerate the individual for subsequent presentation of that antigen.

2. Treatment of Chronic Inflammatory Disease

Since LAD patients lacking LFA-1 do not initiate an inflammatory response, the antagonism of the natural ligand of LFA-1 also appears to inhibit the inflammatory response. The ability of antibodies to ICAM-3 to inhibit inflammation can be attributed to autologous lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of diabetic disease, Raynaud's syndrome, rheumatoid arthritis, etc. It is the basis for therapeutic use in the treatment of immune diseases and chronic inflammatory diseases. Such antibodies may also be used as therapeutics in treating psoriasis.

In general, the anti-ICAM-3 antibodies of the invention can be administered alone or in combination with anti-ICAM-1, anti-ICAM-2 antibodies, or both, for the treatment of diseases currently treatable through steroid treatment. .

In accordance with the present invention, the inflammatory and immune rejection response may be suppressed (ie prevented or attenuated) by providing a patient in need thereof with an amount of anti-inflammatory agent sufficient to suppress the inflammation. Suitable anti-inflammatory agents include antibodies that can bind ICAM-3; A fragment of the antibody capable of binding ICAM-3; Near pure ICAM-3; Functional derivatives of ICAM-3; Non-immunoglobulin antagonists of ICAM-3 or non-immunoglobulin antagonists of ICAM-3 other than LFA-1. Especially preferred are anti-inflammatory agents consisting of soluble functional derivatives of ICAM-3. Such anti-inflammatory treatments include antibodies that can bind LFA-1; Non-immunoglobulin antagonists of LFA-1; And additionally administering an agent selected from the group consisting of substantially pure ICAM-1 and / or ICAM-2 or derivatives thereof, or anti-ICAM-1 and / or anti-ICAM-2 antibodies or fragments thereof.

The present invention also includes the aforementioned method of suppressing the inflammatory response of a specific protective system, wherein an immunosuppressive agent is further provided to the patient. Such formulations are usually preferably provided in amounts lower than the required amount (ie, "below the optimum dose"). Because of the synergistic effect of the formulations of the invention, it is also possible to use doses below the optimum dose. Examples of suitable immunosuppressive agents include, but are not limited to, dexamethasone, azathioprine, ICAM-1, ICAM-2 or cyclosporin A.

3. Treatment of nonspecific inflammation

Many inflammatory responses are due to the response of the "nonspecific defense system" and are mediated by leukocytes without immune memory. Such cells include granulocytes and macrophages. As used herein, “inflammation” is referred to as being caused by a response of a nonspecific defense system, if it is caused, mediated, or related by a response of a nonspecific defense system. Inflammation resulting at least in part from the response of a nonspecific defense system includes adult respiratory distress syndrome (ARDS) or multiple organ damage syndrome dependent on sepsis, trauma or bleeding; Reperfusion injury of the myocardium or other tissues; Acute glomerulonephritis; Reactive arthritis; Skin diseases with acute inflammatory sites; Acute purulent meningitis or other central nervous system inflammatory disease (eg, seizures); Thermal damage; Hemodialysis; Leukocyte clearing blood transfusion; Ulcerative colitis; Crohn 's disease; Necrotizing enterocolitis; Granulocyte transfusion related syndrome; And inflammation associated with conditions such as cytokine-induced toxicity.

Anti-inflammatory agents of the present invention are compounds that can specifically antagonize the interaction of endothelial cells with granulocyte-like CD-18 complexes. Such antagonists include ICAM-3; Functional derivatives of ICAM-3; In addition to members of the ICAM-1, ICAM-2, or CD-18 molecular family, non-immunoglobulin antagonists of ICAM-3 are included.

B. Inhibitors of organ and tissue rejection

In particular, soluble ICAM-3 can act in the same way as antibodies against members of the CD-18 family of molecules, and thus can be used to suppress organ or tissue rejection responses caused by cell attachment-dependent actions. In addition, anti-ICAM-3 antibodies, functional derivatives of ICAM-3 and antagonists of ICAM-3 can also be used to suppress the rejection response.

ICAM-3 and anti-ICAM-3 antibodies prevent solid organ or tissue rejection (e.g., kidney), non-solid organ rejection (e.g., bone marrow) or, in mammalian patients, modify the autoimmune response. It can be used to It is important that organ transplantation can be performed between individuals with HLA mismatches by using monoclonal antibodies capable of recognizing ICAM-3.

C. Adjuvants for the introduction of antigenic substances administered for therapeutic or diagnostic purposes

Immune responses to therapeutic or diagnostic agents, such as bovine insulin, interferon, tissue-type plasminogen activator or mouse monoclonal antibodies, impair the therapeutic or diagnostic effect of such agents, and in some cases, diseases such as serum diseases Cause. Such symptoms can be cured by using the antibodies of the present invention. In an embodiment of the invention, the anti-ICAM-3 antibody is administered in combination with a therapeutic or diagnostic agent. Addition of the antibody prevents the recipient from recognizing the agent, thus preventing the recipient from initiating an immune response against it. The absence of this immune response allows the patient to receive additional therapeutic or diagnostic agents.

ICAM-3 (particularly soluble) or functional derivatives thereof may be used alone or in combination with antibodies capable of binding ICAM-1 or ICAM-2 or LFA-1. Thus, the molecules in dissolved form may be used to inhibit organ or transplant rejection. ICAM-3 or a functional derivative thereof can be used in the same way as an anti-ICAM-3 antibody to reduce the immunogenicity of a therapeutic or diagnostic agent.

D. Suppressors of Tumor Metastasis

The formulations of the invention can also be used to suppress metastasis of hematopoietic tumor cells, which require a functional member of the CD-18 family for migration. According to this embodiment of the invention, an agent sufficient to inhibit said metastasis to a patient in need of said treatment (eg, an antibody capable of binding ICAM-3; a toxin derivative of said antibody; binding to ICAM-3) Fragments of said antibody; toxin derivatives of said fragments; nearly pure ICAM-3; functional derivatives of ICAM-3; or non-immunoglobulin antagonists of ICAM-3 in addition to ICAM-1 or ICAM-2) To provide.

In a further embodiment of the invention, a method of inhibiting the growth of ICAM-3 expressing tumor cells is provided. In particular, the method comprises providing the patient in need thereof with an amount of preparation sufficient to suppress the growth. Suitable agents include antibodies that can bind ICAM-3; Toxin derivatives of the above antibodies; Fragments of such antibodies capable of binding ICAM-3; Toxin derivatives of the fragments; Toxin derivatives of CD-18 molecular family; Or toxin derived functional derivatives of members of the CD-18 molecular family.

According to another embodiment of the present invention, a method of suppressing the growth of LFA-1 expressing tumor cells is provided. In particular, the method comprises providing to the patient in need thereof a sufficient amount of toxin to inhibit the growth. Suitable toxins include toxin derived ICAM-3 or toxin derived functional derivatives of ICAM-3.

E. Repression of HIV Infection and Prevention of Propagation of HIV Infected Cells

In a further embodiment of the invention, a method of suppressing an infection of HIV is provided. In particular, the method comprises administering to the HIV-infected individual an effective amount of an HIV infection inhibitor. Although the present invention relates in particular to a method for suppressing HIV-1 infection, the method also applies to any HIV variant capable of infecting cells (eg HIV-2) in a manner that can be suppressed by the agent of the invention. Can be applied. Such variants are treated equivalent to HIV-1 for the purposes of the present invention.

One aspect of the invention is that transduction of LFA-1 and in some cases binding ligands of LFA-1 stimulated by HIV infection promotes cell-cell adhesion responses that can increase the contact time of infection with uninfected cells, thereby infecting cells. Derives from the recognition that it can facilitate viral transfer from the infected to uninfected cells. Thus, formulations of the present invention capable of modulating LFA-1 / ligand interactions can suppress infection by HIV and in particular HIV-1. One way that molecules that inhibit LFA-1 / ligand interactions can suppress HIV infection is that LFA-1 ligands expressed by HIV-infected cells can bind to CD11 / CD18 receptors in healthy T cells. It is impairing ability. Alternatively, molecules capable of inhibiting LFA-1 / ligand interactions may impair the ability of CD11 / CD18 receptors expressed by HIV-infected cells to bind LFA-1 in healthy T cells. . To impair the ability of cells to bind to CD11a / CD18 receptors or LFA-1 binding ligands, ICAM-3, fragments of ICAM-3, functional derivatives of ICAM-3 or anti-ICAM-3 antibodies are used. It is possible to do

The formulations of the present invention are provided to recipients in an amount sufficient to suppress HIV infection. This amount is said to be sufficient to "suppress" the HIV infection if the dosage, route of administration, etc. of the formulation is sufficient to attenuate or prevent HIV infection. This formulation is given to patients exposed to or affected by HIV infection.

The formulations of the present invention can be used for "prophylaxis" or "therapeutic" in the treatment of HIV infection. When provided for prophylaxis, the agent is given prior to the symptoms of viral infection (eg, before, during or immediately after infection). Prophylactic administration of the agent serves to prevent or attenuate subsequent HIV infection. When provided for treatment, the agent is provided upon detection of (or immediately after) a virus infected cell. Therapeutic administration of the agent then serves to attenuate HIV infection.

Thus, the formulations of the present invention may be provided before the onset of viral infection (for the purpose of inhibiting HIV infection foreseen) or after the actual detection of virally infected cells (for the purpose of inhibiting subsequent infection).

In another aspect, the present invention provides improved therapies for AIDS, and improved means for suppressing HIV infection, in particular HIV-1 infection, comprising: (I) ICAM-3, soluble ICAM-3 derivatives, CD11 ( CD11a, CD11b or CD11c), soluble CD11 derivative, CD18, soluble CD18 derivative, or CD11 / CD18 heterodimer, or soluble derivative of CD11 / CD18 heterodimer, and / or (II) an antibody capable of binding to ICAM-3, Co-administering (III) CD4 or a soluble derivative of CD4 associated with the cell or particle, and / or (IV) a molecule capable of binding to CD4 (preferably an antibody or antibody fragment).

In another aspect of the invention, a method of suppressing migration of HIV-infected cells is provided. Specifically, the method comprises administering an effective amount of an anti-migrating agent to an HIV-infected individual.

Anti-migration agents of the invention include ICAM-3, fragments of ICAM-3, functional derivatives of ICAM-3, or anti-ICAM that can impair the ability of HIV-infected T cells to bind LFA-1 ligands. -3 antibodies. Antibodies that bind ICAM-3 will suppress migration by impairing the ability of ICAM-3 expressed by HIV-infected T cells to bind to cells expressing the CD11 / CD18 receptor. In order to impair the ability of cells to bind to CD11a / CD18 receptors, it is possible to use antibodies capable of binding to ICAM-3.

The formulations of the present invention should be provided to recipient patients in an amount sufficient to suppress the migration of T cells infected with HIV (or other viruses). If the dosage, route of administration, etc. of the agent is sufficient to attenuate or hinder such migration, it is said to be sufficient to "suppress" the migration of T cells.

Administration of such compounds may be aimed at "prophylaxis" or "treatment". When provided for prophylaxis, the formulations of the present invention are provided before any symptoms of a viral infection appear (e.g., before, at or immediately after the infection, but before any symptoms of the infection appear). do. Prophylactic administration of the agent serves to prevent or attenuate any subsequent migration of virus infected T cells. When provided for therapeutic use, the agent is provided upon detection of (or immediately after) the virus infected T cells. Therapeutic administration of the agent serves to attenuate any additional migration of the T cells.

Thus, the formulations of the present invention may be provided before the onset of virus infection (to suppress the expected migration of infected T cells) or after substantial detection of the virus infected cells.

F. Asthma Treatment

In another embodiment of the invention, agents capable of modulating LFA-1 / ICAM-3 interactions are used for the treatment of asthma. Specifically, this method involves administering an effective amount of an anti-asthma agent to a patient in need of this treatment.

Anti-asthmatic agents of the invention include ICAM-3, ICAM-3 fragments, functional derivatives of ICAM-3, or anti-ICAM-3 antibodies that can impair the ability of cells to bind LFA-1. Antibodies that bind ICAM-3 will suppress eosinophil migration by impairing the ability of ICAM-3 expressed in eosinophils to bind to cells expressing the CD11 / CD18 receptor.

The anti-asthmatic formulations of the present invention should be provided to a receiving patient in an amount sufficient to attenuate the depth, severity or duration of asthma symptoms.

The formulations of the invention may be used alone or in combination with one or more additional anti-asthmatic agents such as methylxanthine (such as theophylline), beta-adrenergic agonists such as catecholamines, resorcinol, salinine and epi Drin), glucocorticoids (eg hydrocortisone), chromones (eg sodium chromoline) and anticholinergic drugs (eg atropine) may be administered together to reduce the amount of the agent required to treat asthma symptoms.

Administration of such compounds may be aimed at "prophylaxis" or "treatment". When provided for prophylaxis, the formulations of the present invention are provided before any symptoms of asthma appear. Prophylactic administration of such agents serves to interfere or attenuate any subsequent asthma reaction. When provided for treatment, the agent is provided at (or immediately after) the onset of asthma symptoms. Therapeutic administration of such agents serves to attenuate any substantial asthma episodes.

Thus, the formulations of the present invention may be provided before the onset of a predicted asthma episode (to attenuate the predicted depth, duration or extent of the episode) or after the start of the episode.

G. Diagnostic and prognostic applications

Monoclonal antibodies capable of binding ICAM-3 can be used as a means of imaging or visualizing ICAM-3 expression and inflammation sites in patients. For such use, anti-ICAM-3 monoclonal antibodies are detectably labeled using radioisotopes, affinity labels (eg, biotin, avidin, etc.), fluorescent labels, or paramagnetic atoms. Methods of carrying out such labeling are well known in the art. The labeled antibody can then be used for diagnostic imaging. The clinical application of antibodies in diagnostic imaging is described in Grosman, H. B. Urol. Clin. North Amer. 13: 465-474 (1986), Unger, E. C. et al. Invest. Radiol. 20: 693-700 (1985) and Koo, B. A. et al., Science 209: 295-297 (1980).

The presence of ICAM-3 expression may also be indicated by hybridization probes, such as mRNA, cDNA, genomic DNA, or synthetic oligonucleotides, which bind to the ICAM-3 gene sequence or to the ICAM-3 mRNA sequence present in cells expressing ICAM-3. Can be detected using a probe. Techniques for conducting such hybridization assays can be found in Molecular Cloning, Manitentis, T. et al., Laboratory Manual, Cold Spring Harbour, New York (1982), and Nucleic Acid Hybridization, Practical Approach, such as Hayes, BD, IRL Press, Washington, DC (1985), incorporated herein by reference.

Detection of ICAM-3 expression sites using labeled antibodies or nucleic acid probes can be used to diagnose tumor development. In one diagnostic embodiment, a tissue or blood sample is removed from a patient and incubated in the presence of an antibody that can be labeled or detectably detectable. In a preferred embodiment this technique is done in a non-invasive way using magnetic imaging, fluorescence photography and the like. Such diagnostic tests can be used to monitor organ transplant receptors (eg kidneys) for early signs of potential tissue rejection. Such an assay may also be performed in an attempt to determine an individual's bias for rheumatoid arthritis or other chronic inflammatory disease.

For example, by radiolabeling an anti-ICAM-3 antibody or antibody fragment, it is possible to detect antigen using radioimmunoassay. A good description of radioimmunoassay (RIA) can be found in "Laboratory Technology and Biochemistry in Molecular Biology, Work, TS et al., North Holland Publishing Company, New York (1978)", in particular "Radioactive Immunoassay and Related Techniques". The chapter entitled "Introduction to E, Chad, T" is hereby incorporated by reference. On the other hand, the antibody can be labeled with fluorescent compounds, enzymes, or other suitable labels known in the art.

In addition to localizing the site of inflammation, antibodies capable of binding to ICAM-3 can be used to analyze biological fluids for the presence of circulating ICAM-3 using one of the media commonly used for body fluid analysis. The presence of circulating ICAM-3 in body fluid samples is an indication of an inflammatory response or other ICAM-3 mediated biological action. In addition, the presence of amniotic fluid ICAM-3 is associated with complications during pregnancy. Any biological fluid can be used for the analysis. Preferred biological fluids, however, are blood, serum, plasma, synovial fluid, amniotic fluid, spinal fluid or urine.

VIII. Administration of the Compositions of the Invention

The therapeutic effect of ICAM-3 can be obtained by providing the patient with the entire ICAM-3 molecule, or any peptide fragment with therapeutic activity thereof. Of particular interest are peptide fragments with therapeutic activity of soluble ICAM-3.

ICAM-3 and its functional derivatives can be obtained by proteolysis or by a combination of such methods by synthesizing using recombinant DNA technology. The therapeutic benefit of ICAM-3 can be increased using functional derivatives of ICAM-3 with additional amino acid residues added to increase binding to the carrier or increase the activity of ICAM-3. The scope of the present invention includes functional derivatives of ICAM-3 that lack certain amino acid residues and have modified amino acid residues, which possess or otherwise influence the biological or pharmaceutical activity of ICAM-3. So far as it is concerned.

Antibodies of the invention and the ICAM-3 molecules described herein can be said to be "almost free of natural contaminants" if there are few substances normally found together naturally with these products in the preparation containing the product.

The invention also includes antibodies capable of binding to ICAM-3, and biologically active fragments thereof (which may be multiple clones or monoclonals). Such antibodies can be produced by animal or tissue culture or DNA recombination.

Molecules derived from ICAM-3, or ICAM-3, can be administered alone or in combination with ICAM-1 or ICAM-2. Anti-ICAM-3 antibodies, or other molecules capable of binding to ICAM-3 or molecules derived from ICAM-3, may be administered alone or with anti-ICAM-1 antibodies, anti-ICAM-2 antibodies, or both. May be administered together. In providing a patient with an antibody or antibody fragment capable of binding to ICAM-3, or when providing a recipient with ICAM-3 (or a fragment, variant or derivative thereof), the dosage of the agent administered is determined by It will depend on factors such as age, weight, height, sex, general medical conditions and prior medical history. In general, a preferred but less than or greater than one dose of antibody ranges from about 1 pg / kg to 10 mg / kg (patient body weight) may be administered. When providing a patient with an ICAM-3 molecule or a functional derivative thereof, it is preferred to administer these molecules at a dosage in the range of about 1 pg / kg to 10 mg / kg (weight of patient), but at lower or higher dosages as well. May be administered. As mentioned above, if an anti-ICAM-3 antibody is administered with an anti-LFA-1 antibody, an anti-ICAM-1 antibody and / or an anti-ICAM-2 antibody, the therapeutically effective amount can be reduced. As used herein, it can be said that one compound is additionally administered with a second compound if the two compounds are administered almost simultaneously such that the two compounds are detected simultaneously in the serum of the patient.

Antibodies capable of binding to ICAM-3 and ICAM-3 itself may be administered to a patient intravenously, intramuscularly, subcutaneously, intestinal, topically or parenterally. When the antibody or ICAM-3 is administered by infusion, it can be administered by continuous infusion or by one or multiple lesions.

The formulations of the present invention will be provided to the recipient sample in an amount sufficient to be "physiologically effective". If the dosage, route of administration, and the like of the agent are sufficient to attenuate or prevent the physiological effects associated with ICAM-3, the amount can be said to be physiologically effective. For example, one of the agents of the present invention that provide a patient to inhibit inflammation can be said to be physiologically effective if provided in a dosage sufficient to “suppress” inflammation.

The anti-ICAM3 antibody, or fragment thereof, may also be administered alone or in combination with one or more additional immunosuppressive agents, particularly to receptors undergoing organ or tissue transplantation. Administration of such compound (s) may be "prophylactic" or "therapeutic". When provided for prophylaxis, the immunosuppressive compound (s) are given before any inflammatory response or symptom occurs (eg, before organ or tissue transplantation, at the time of transplantation or shortly after transplantation, but without organ rejection). May be given immediately before symptoms appear). Prophylactic administration of the compound (s) prevents or attenuates any subsequent inflammatory response (eg, rejection of transplanted organs or tissues, etc.). When provided for therapeutic use, the compound (s) are given when an actual inflammatory condition (eg, organ or tissue rejection) develops (immediately after the onset). The therapeutic dose of the compound (s) attenuates any actual inflammation (eg, rejection of transplanted solid organs or tissues such as kidneys, or non-solid organs such as bone marrow).

The anti-inflammatory agents of the present invention may therefore be provided before the onset of inflammation (to suppress the expected inflammation) or after the onset of inflammation. If the administration of a composition is tolerable for a receiving patient, the composition may be said to be “pharmaceutically acceptable”. If the amount to which such agent is administered is physiologically significant, it can be said to be administered in a "therapeutically effective amount". Physiologically important if the administration of such agents results in a detectable change in the physiology of the recipient patient.

The antibodies and ICAM-3 molecules of the present invention can be formulated according to known methods for preparing pharmaceutically useful compositions, whereby these substances or functional derivatives thereof can be mixed with pharmaceutically acceptable carrier excipients. Suitable excipients and other preparations comprising other human proteins such as human serum albumin are described, for example, in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A., Ed., Mac, Eastern PA (1980). Described. In order to form a pharmaceutically acceptable composition suitable for effective administration, the compositions will contain an effective amount of the formulation of the present invention together with an appropriate amount of carrier. In addition, the antibodies of the invention can be humanized by chimerization or CDR transplantation to make them more "pharmaceutically acceptable" to the patient. Methods for chimerizing antibodies, in particular anti-ICAM-1 antibodies, include, in addition to the above documents, British Patent Application Nos. 9009548.0 and 9009549.8 and PCT Application Nos. PCT / US91, filed April 27, 1991, to the U.S. Repair Office. / 02942 and PCT / US91 / 02946, which are incorporated herein by reference.

Additional pharmaceutical methods can be used to control the duration of action. Controlled release formulations can be obtained by using polymers for incorporating or absorbing the formulations of the invention. The controlled delivery rate and delivery duration can be controlled to some extent by selecting a suitable macromolecular matrix, changing the concentration of macromolecules incorporated, and the method of incorporation. Another method for controlling the duration of action by controlled release formulations is to incorporate the formulations of the invention into particles of a polymeric material such as polyester, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinyl acetate copolymers. will be. Instead of incorporating these agents into the polymer particles, these materials can also be prepared, for example, by coacervation techniques or by interfacial polymerization, or by microcapsules prepared by gelatin or poly (methylmethacrylate) microcapsules. Or, for example, entrapped in a colloidal drug delivery system such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or large emulsions.

The invention also relates to (a) an anti-inflammatory agent (an antibody capable of binding to ICAM-3; a fragment of said antibody capable of binding to ICAM-3; ICAM-3; a functional derivative of ICAM-3; or ICAM-1 and Non-immunoglobulin antagonists of ICAM-3 other than ICAM-2), and (b) one or more immunosuppressive agents. Examples of suitable immunosuppressive agents include dexamethasone, azathioprine and cyclosporin A. The formulations of the present invention and methods of obtaining the same will now be more readily understood by reference to the following examples, which have been described above generally and are not intended to limit the invention but to specifically illustrate it.

Example 1

Characterization of ICAM-3, a Novel Attachment Ligand for LFA-1

Attachment of cell lines and lymphocytes to LFA-1-coated plates (Dustin et al., Cold Spring Harbor Symp. Quant. Biol. 54: 753-765 (1989)) was performed as previously described. LFA-1 purified from JY lysate was adsorbed at 1100 sites / μm ² on 96 well microtiter plates (Linbro-titertek). Non-specific binding sites were blocked with 1% BSA and washed with PBS / 5% FBS / 2mM MgCl ₂ /0.5% HSA (analyte). Specific inhibition of LFA-1 was obtained by incubating the microtiter wells with 1/200 dilution of TS1 / 22 (anti-LFA-1α) ascites for 30 min at RT. Resting T cells were isolated from total blood by plastic adhesion and nylon wool filtration and were 91% CD2 ⁺ , whereas PHA-blasts were produced by culturing cells in the presence of 10 μg / ml phytohemagglutinin (PHA). It became. Cells were labeled with fluorescent dye BCECF (Molecular Probes Inc.), washed and resuspended in assay solution. MAb pretreatment of cells was done by transferring 10 ⁵ cells to each well and then incubating for 45 min at 1/200 plural dilution at 4 ° C. Cell lines were attached to solid LFA-1 for 1 hour at 37 ° C., and non-adherent cells were removed by six 23 gauge needle aspirations, and lymphocytes were spun at 30 × g for 5 minutes and then at 37 ° C. for 30 minutes. It was incubated. Unbound lymphocytes were removed by tapping 8 times with medium from each plate with 100 ψ added between washes. By tapping the plate, it was more effective at thoroughly removing unbound T lymphocytes, which were smaller and more difficult to remove. Fluorescence was quantified directly from 96 well plates by using a Fluorescent Concentration Analyzer (Baxte). All MAbs were used at saturation concentrations (1/200 plural dilutions) and TS1 / 22 (anti-CD 11a, IgG1) (Sanches-Madrid et al., Proc. Natl. Acad. Sci. USA 79: 7489-7493 (1982) )), RR 1/2 (anti-ICAM-1, IgG1) (Rodlein et al., J. Immunol. 137: 1270-1274 (1986)), CBR-IC 2/2 (anti-ICAM-2, IgG2a) ) (Defugerrows et al., J. Exp. Med. Submitted (1991) (published)), and CBR-IC 3/1 (anti-ICAM-3, IgG1). CBR-IC 3/1 was induced by the fusion of splenic cells from SKW 3-injected Balb / c mice with rat myeloma P3Ⅹ63Ag8.653 (Gefter et al., Som. Cell Gen. 3: 231-236 (1977). )), 600 hybridomas were searched for their ability to inhibit SKW3 binding to purified LFA-1. CRB-IC 3/1 did not respond to purified LFA-1 and did not respond to COS cells transfected with ICAM-1, ICAM-2 or LFA-1 cDNA (data not shown). One of four representative experiments is shown and error bars represent one standard deviation.

Example II

Flow cytometry assay of ICAM-1, ICAM-2, and ICAM-3 expression of cells

All cell lines used are described in Hips et al., J. Clin. Invest. 85: 674-681 (1990). Peripheral blood mononuclear cells (PBMCs) were obtained by dextran sedimentation and Ficoll-Hypaque (1.077) centrifugation, which were described in Dustin et al., J. Cell. Biol. 107: 321-331 (1988). Neutrophils were recovered from cell pellets and contaminant red blood cells were removed by hypotonic lysis. Lymphocytes and monocytes were isolated by cytometric analysis using anterior and vertical light scatterers and identified by monocytes and T cell specific MAbs. Plastic-adhesive was used to enrich lymphocytes in PBMCs and cells were cultured in the presence of 10 μg / ml phytohemagglutinin (PHA). Samples were analyzed using EPICS V flow cytometer and fluorescence was quantitated using EPICS Immune-Brite fluorescent beads (Coulter) to calibrate the cytometer. Expression of ICAM-3 on resting lymphocytes was 2-3 times greater than CD3 or LFA-1 and monocytes expressed 3-4 times more ICAM-3 than LFA-1. The expression level of ICAM-3 on neutrophils was the same as that of Mac-1 (CD11b / CD 18). Treatment of cells with phospholipase C showed no PI-linked form of ICAM-3 present.

Example III

Immunoprecipitation of ICAM-3

Surface labeling of cells by ¹²⁵ I has been described using Iodogen, Kishimoto et al., J. Biol. Chem. 264: 3588 (1989). Cells were lysed with Triton × -100 (1%). This lysate was prewashed with bovine IgG bound-sepharose and then incubated with the cut MAb-bound sepharose for 2 hours. The beads were washed and heat treated in sample buffer containing 50 mM Tris, 1% SDS and 1% 2-mercaptoethanol. The unreduced sample was heat treated in a buffer lacking 2-mercaptoethanol and then treated with 20 mM iodine acetamide. Samples were analyzed on a 7% vertical slab polyacrylamide gel as described in Laemry, UK, Nature 227: 680-685 (1970) and the proteins visualized by autoradio photography. Treatment of the sample with N-glycanase (Genzyme) using concentrations (10 units / ml, 37 ° C., 18 h) as described in Tarentino et al., Biochemistry 24: 4665-4671 (1985) was performed. It was determined to be optimal for cleaving all of the N-linked oligosaccharides in the backbone. The MHC I group includes N-linked carbohydrates, while ICAM-2 (m _r 60,000, 6 N-binding sites, Mr 28,383 of the backbone) is highly sugarified.

Example IV

Distribution of ICAM-3

Anti-ICAM-3 antibodies of the invention were used to measure the intracellular distribution of ICAM-3 protein using a flow cytometer. Table 3 summarizes the cell types exhibiting surface expression of ICAM-3. Expression of ICAM-3 appears to be limited to hematopoietic cells. Flow cytometry data was confirmed by performing immunohistochemical staining of tissue sections using labeled anti-ICAM-3 antibodies. As in flow cytometry data, immunohistochemistry studies demonstrated that ICAM-3 expression was limited to hematopoietic cells.

Example V

Purification of ICAM-3

ICAM-3 was purified by using immunoaffinity chromatography in which the anti-ICAM-3 antibody CBR-IC 3/1 was immobilized on the matrix by methods known in the art. Numerous sources have been used as starting materials for separating ICAM-3. This starting material includes, but is not limited to, SKW3, human thymoma cell lines, and human tonsils. The method used to purify ICAM-3 is almost as described in J. Exp. Med. 175: 185-190 (1992). Those skilled in the art will appreciate that the washing and elution reagents vary slightly for each antigen to be purified and for each antibody to be used for immunoaffinity chromatography.

As noted below, ICAM-3 purified from SKW3 cells or human tonsil cells appears to have a molecular weight of about 120-124 kD, whereas ICAM-3 purified from lysates derived from neutrophils is about 120-150 kD. The wide band which shows the molecular weight of was computed.

ICAM-3 purified in this manner has been found to retain anti-ICAM-3 antibodies, and the ability to bind LFA-1 on a variety of cells (FIGS. 6 and 7).

Example VI

Identification of ICAM-3 of Various Molecular Weights

Analysis by immunoprecipitation and polyacrylamide gel electrophoresis was used to determine the molecular weight of ICAM-3 in various cell types. The molecular weight of ICAM-3 immunoprecipitated from neutrophils was found to be different from that of immunoprecipitated from lymphocytes. ICAM-3 isolated from lymphocytes and lymphocyte-like cell lines appear as bands having a molecular weight of about 120-124 kD. The molecular weight of ICAM-3 isolated from neutrophils was somewhat higher than that expressed by lymphocyte-like cells and appeared as a diffusion band of about 120 kD to 150 kD (FIG. 4).

Such size changes are common in members of the ICAM family of glycoproteins. ICAM-1 shows similar variations in molecular weight between different cell types. The change in molecular weight seen in ICAM-1 was found to be caused by a change in the degree of sugar addition. Therefore, the change in the molecular weight of ICAM-3 is most likely caused by the difference in sugar addition. One skilled in the art can readily identify ICAM-3 isolated from neutrophils by treating various glycosidase and other enzymes and observing the effect on molecular weight.

Changes in the degree of sugar addition of ICAM-1 were found to affect MAC-1 (CD 11b / CD 18) binding. Thus, by varying the degree of sugar addition of ICAM-3, it is possible to produce ICAM-3 derivatives with modified affinity that bind to various ligands of ICAM-3, for example, members of the CD11 / CD18 family of glycoproteins. .

Example VII

ICAM-3 Antibody

Mice were injected with a mixture of ICAM-3 protein and adjuvant purified from SKW3 or tonsil cells as described above. Monoclonal antibodies of immunized animals were generated using procedures known in the art.

Table 4 summarizes the various anti-ICAM-3 antibodies obtained. Multiple antibodies have been identified based on their ability to respond to immobilized ICAM-3 purified by ELISA. Positive mAbs were detected on various cell lines known to be ICAM-3 positive and then immunoprecipitated in radiolabeled ICAM-3 positive cell lysates. mAbs were also tested for their ability to block PMA-induced SKW3 cell aggregation in the presence of anti-ICAM-1 and anti ICAM-2 antibodies. In addition, anti-ICAM-3 antibodies can be identified by their ability to inhibit binding of SKW3 cells to immobilized purified ICAM-3.

One of the evidences that there is a third ligand of LFA-1 is the presence of an ICAM-1 / ICAM-22 independent pathway for PMA-stimulated SKW3 cell aggregation. We could block this aggregation with mouse anti-ICAM-3 polyclonal antiserum or a mixture of CBR-IC 3/1 and CBR-IC 3/2.

Table 4ICAM-3 mAbs mAb genotype PMA-derived Cohesion ^* Exclusive + IC1 + 2 mAb + 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 antisera 3 0 0 CBR-IC3 / 1 IgG1, k 5 4 - HP2 / 19 ^** One * J. J. Exp. Med. Agglomeration size of the 1991 paper] 0 = non-agglomerate ** Another ICAM-3 mAb obtained from a laboratory in Spain (F. Sanchez-Madrid). After presentation of the results, HP2 / 19 were manufactured but not characterized. One of the mAbs was recalled. It turned out to be ICAM-3. CBR-IC3 / 2, 3/3, 3/5 immunoprecipitates the same 120 kD band as CBR-IC 3/1. Test mAbs for ability to Western blot.

Example VIII

Immunological Analysis Using Anti-ICAM-3 Antibodies

Mixture of anti-ICAM-1 antibody (RR 1/2), anti-ICAM-2 antibody (CBR-IC 2/2) and anti-ICAM-3 antibody (CBR-IC 3/1 and CBR-IC 3/2 ) (1) PMA-stimulated SKW3 cell adhesion to purified ICAM; (2) stimulation of phytohemagglutinin (PHA) against cell division, and (3) the ability to block mixed lymphocyte responses (MLR).

It has been previously found that PMA can activate LFA-1, thereby increasing the ability to bind ICAM-1 (Dusten et al., Network 341: 619 (1989)). 5 shows that similar activation mechanisms are present at the LFA-1 / ICAM-3 binding.

The ability of various antibodies to block binding of PMA stimulated SKW3 cells to purified ICAM-1 and ICAM-3 was investigated (FIG. 6). The presence of antibodies CBR-IC 3/1 and CBR-IC 3/2 alone does not effectively block binding of PMA stimulated SKW3 cells to purified ICAM-3. However, PMA stimulated SKW3 cell binding to ICAM-3 can be blocked when using a mixture of the two antibodies. See J. Exp. Med.], CBR-IC3 / 1 monoclonal antibody itself could block the binding of ICAM-3 to purified LFA-1.

Binding of PMA stimulated SKW3 cells to ICAM-3 was further analyzed to determine whether it was temperature dependent or cation dependent. As in ICAM-1, ICAM-3 / LFA-1 binding was found to be temperature dependent (FIG. 8) and cation dependent (data not shown).

9 summarizes the effects of various antibodies on PHA stimulation of cell division. As is known in the art (Krensky et al., J. Immunol. 131: 611-616 (1983)), PHA stimulates cell proliferation in a manner that can be inhibited with anti-LFA-1 in anti-ICAM-1 antibodies. , Anti-ICAM-2 antibodies, or mixtures of anti-ICAM-1 and anti-ICAM-2 antibodies had little effect on PHA stimulated cell division. However, 1) anti-ICAM1, anti-ICAM-2, and anti-ICAM-3 antibodies, 2) two anti-ICAM-3 antibodies (IC3 / 1 and IC3 / 2), and 3) anti-ICAM-1 and Mixtures of anti-ICAM-3 antibodies were effective at blocking PHA stimulated cell division.

Example IX

Mixed lymphocyte response

MLR assays are used to assess immunological reactivity. The assay basically involves adding chemically immobilized foreign cells (stimulatory cells) to a solution containing PBLs from several individuals (reactive cells) and measuring the degree of reactivity using the proliferation of the reactive cells as an indicator. And conventionally known [Krensky et al., J. Immunol. 131: 611-616 (1983). This assay is the first step in identifying compositions useful for treating transplant rejection.

The effect of various antibodies and antibody mixtures on the MLR response is shown in FIG. 10. In summary, the use of anti-ICAM-3 alone showed a low effect. However, the use of anti-ICAM-1 and anti-ICAM-3 antibody mixtures have been shown to have a great effect on blocking MLR. The highest response obtained was when using a mixture of anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3 antibodies.

Example X

Peptide Sequences of ICAM-3

ICAM-3 was purified from human tonsil cells by immunoaffinity chromatography as described in Example 5. Purified ICAM-3 was enzymatically cleaved with Lys-C enzymes by known methods and peptide fragments were separated by HPLC. Figure 11 shows the various chromatographic peaks observed in the general digestion by gas chromatography. Peaks 10 and 17 were identified as containing peptide fragments of sufficient size and structure to be sequenced (hereinafter named NK-10 and NK-17 peptides, respectively).

The amino acid sequences of the NK-17 and NK-10 peptides were determined using standard procedures. The sequence of NK-10 protein was found to be KIDRATCPQHLK (SEQ ID NO: 1). The first lysine (K) residue indicated in this sequence was presumed to exist because Lys-C cleaved the protein following the lysine residue.

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

The comparison of the known ICAM-1 and ICAM-2 sequences with the NK-10 and NK-17 proteins showed high homology. NK-17 peptides show significant homology to sequences contained within the first Ig domain of ICAM-2. NK-10 peptides show weak homology to sequences in domain 4 of ICAM-1.

Example XI

CDNA cloning of ICAM-3

Degenerate oligonucleotide probes were generated based on the peptide sequences obtained above. The probe was further designed to include previously identified codon preferences observed in ICAM-1 and ICAM-2 nucleotide sequences. The nucleotide sequence of the probe based on the amino acid sequences of the NK-17 and NK-10 proteins is as follows:

Oligo 23 coalescence with 24x degeneracy degree containing NK-17 probe, 2N (inosine).

NK-10 probe. 25 coalesces with an 8-fold degree of degradation containing 5N (inosine).

cDNA transduction libraries were generated from amygdala RNA as described above (June et al., Proc. Natl. Acad. Sci US 82: 7711-7716 (1985)). The library was searched using the NK-17 probe and plaques showing positive hybridization were rescanned using the NK-10 probe. One clone, clone 11.2, was found to have an insert between 1.6 and 1.8 kb in length. A schematic representation of clone 11.2 and the positions of the NK-10 and NK-17 peptides are shown in FIG.

The ends of the inserts were sequenced by procedures known to those skilled in the art using primers derived from adjacent vectors, whereas the NK-10 probe was used as a primer for internal sequencing.

Shown in FIG. 18 summarizes the sequences obtained above. The sequence reveals many facts. First, sequences with high homology with the transmembrane region of ICAM-1 were identified. Thus, if one wishes to produce a soluble fragment of ICAM-3, one will have to delete all sequences after the start of the transmembrane region shown in FIG. Second, the sequence obtained at the 5 'end of clone 11.2 is likely to be close to the 5' end of a naturally occurring gene, as presumed by sequence homology between ICAM-1, ICAM-2 and ICAM-3. .

Example XII

Sequence homology between different ICAM molecules

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

The results show that ICAM-3 shows high homology with ICAM-1 and ICAM-2. Sequence homology between ICAM-1 and ICAM-3, as well as some of the transmembrane and cytoplasmic domains of ICAM-1 (FIGS. 13 and 14), as well as the first domain (FIG. 16) and 4/5 of ICAM-1 Found in the domain. Further search of the database of the gene bank revealed no other sequences identical or similar to those obtained in ICAM-3. Significant homology was observed between ICAM-3 and the first domain of ICAM-2 (FIG. 17). Clone 11.2 was used to search the library further. Additional clones were searched to obtain cDNA inserts of about 2 to 2.4 kD. Larger sizes have not been identified to date, so it is assumed that they represent cDNA clones of total length.

The present invention provides an intercellular adhesion molecule ICAM-3, a method for identifying ICAM-3, a method for treating inflammation using ICAM-3 and antibodies that can bind to them, and for treating diseases such as AIDS. Provide a method.

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Claims (62)

a) the amino acid sequence of FIG. 18A; And b) amino acid sequence of FIG. 18B An ICAM-3 fragment having a deletion of a transmembrane region comprising at least one amino acid sequence selected from the group consisting of: The fragment of claim 1 selected from the group consisting of SEQ ID NOs: 2 and 4. 3. a) the amino acid sequence of FIG. 18A; And b) amino acid sequence of FIG. 18B A recombinant nucleic acid molecule comprising a nucleotide sequence encoding at least one amino acid sequence selected from the group consisting of, and capable of encoding an ICAM-3 fragment in which a transmembrane region is deleted. 4. The recombinant nucleic acid molecule of claim 3, wherein the transmembrane region can encode an ICAM-3 fragment, wherein the fragment is selected from the group consisting of SEQ ID NOs: 1 and 3. The recombinant nucleic acid molecule of claim 3, wherein the nucleotide sequence encodes the amino acid sequence of FIGS. 18A and 18B. The method of claim 3, wherein the nucleic acid molecule is a) the nucleotide sequence of Figure 18A; And b) nucleotide sequence of FIG. 18B Recombinant nucleic acid molecule comprising at least one nucleotide sequence selected from the group consisting of. A biological agent comprising an effective amount of an ICAM-3 modulator and modulating a cell's ICAM-3 mediated biological action, wherein the ICAM-3 modulator comprises: an ICAM-3 fragment lacking a transmembrane region; An antibody capable of binding to ICAM-3 in which the transmembrane region is deleted; A biological agent selected from the group consisting of fragments of said antibody capable of binding ICAM-3 having a deleted transmembrane region. A biological agent comprising an effective amount of an HIV-1 infection inhibitor and inhibiting infection of leukocytes by HIV, wherein the inhibitor comprises an ICAM-3 fragment lacking a transmembrane region; An antibody capable of binding to ICAM-3 in which the transmembrane region is deleted; Toxin derivatives of this antibody; A fragment of the antibody capable of binding ICAM-3 with a deletion of the transmembrane region; Biological agent selected from the group consisting of toxin derivatives of the fragment. A biological agent containing a sufficient amount of toxins to inhibit proliferation of ICAM-3 expressing tumor cells and inhibiting proliferation of ICAM-3 expressing tumor cells, wherein the toxins are present in ICAM-3 lacking a transmembrane region. Antibodies capable of binding; Toxin derivatives of this antibody; A fragment of the antibody capable of binding ICAM-3 with a deletion of the transmembrane region; Biological agent selected from the group consisting of toxin derivatives of the fragment. A biological agent comprising a sufficient amount of toxin to inhibit proliferation of LFA-1 expressing tumor cells, wherein the toxin is a toxin-induced, transmembrane region-deleting ICAM. -3 biological agents. ICAM-3 fragments with deletions of transmembrane regions; An antibody capable of binding to ICAM-3 in which the transmembrane region is deleted; A biological agent comprising an agent selected from the group consisting of fragments of antibodies capable of binding ICAM-3 deleted from the transmembrane region. A diagnostic composition for detecting ICAM-3 comprising an antibody molecule or fragment thereof that is capable of binding to ICAM-3 having a deletion of a transmembrane region and is detectably labeled. A method of identifying an agent capable of modulating binding of ICAM-3 to LFA-1, in which the diaphragm region is deleted, comprising the steps of contacting ICAM-3, in which the diaphragm region is deleted, with LFA-1 in the presence of the agent, and Measuring the modulation of the binding. (a) incubating the cell or cell extract in the presence of a nucleic acid molecule capable of hybridizing to mRNA for ICAM-3 in which the transmembrane region is deleted under stringent conditions; And (b) detecting the presence of cells expressing ICAM-3 lacking a transmembrane region, comprising determining whether the nucleic acid molecule has hybridized to the cell or to a complementary nucleic acid molecule present in the extract of the cell. How to. (a) incubating a tissue sample of a mammalian patient with a composition comprising an antibody, or fragment thereof, capable of binding to a cell expressing ICAM-3 lacking a transmembrane region; And (b) detecting the expression of ICAM-3 deleted in the transmembrane region, comprising detecting the antibody bound to the cell. (a) incubating a biological fluid sample of a mammalian patient with a composition comprising an antibody, or fragment thereof, capable of binding ICAM-3 having a deleted transmembrane region; And (b) detecting the presence of ICAM-3 in which the diaphragm region is deleted in a bodily fluid sample taken from a mammalian patient, comprising detecting the antibody bound to the bodily fluid. The method of claim 16, The body fluid is urine; The method of claim 13, And the ICAM-3 having the LFA-1 and the transmembrane region deleted is purified to be free of contaminants present naturally. The method of claim 13, Wherein said LFA-1 is expressed on a cell surface and purified such that there is no naturally present contaminant of said ICAM-3, which has deleted a transmembrane region. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete