DK176020B1 - Inter-cellular adhesion molecules - used for producing antibodies for use as antiinflammatory agents, to modify immune responses or as antitumour agents - Google Patents

Abstract

Intercellular Adhesion Molecule-1 (CAM-1) or a functional deriv. free of natural contaminants, is claimed. Also claimed is a recombinant DNA molecule capable of expressing ICAM-1 or a functional deriv. and antibodies capable of binding ICAM-1 or a functional deriv. Also claimed is a method of identifying a non-immunoglobulin antagonist of intercellular adhesion which comprises (a) incubating a non-immunoglobulin agent with a lymphocyte prepn. contg. cells capable of aggregating, (b) examining the lymphocyte prepn. to determine whether the presence of the agent inhibits the aggregation of the cells.

Description

in DK 176020 B1

The present invention relates to monoclonal antibodies as well as fragments thereof capable of binding to intercellular adhesion molecules. The invention further relates to cells for producing such antibodies. In another aspect, the invention relates to a method for diagnosing tumor cells expressing intercellular adhesion molecules, a pharmaceutical composition containing the antibodies or fragments thereof, and the use of the antibodies or fragments to prepare an antibody. pharmaceutical composition for the treatment of inflammation or suppression of tumor growth.

Leukocytes must be able to attach to cell substrates to properly defend the host 151 against foreign invaders, such as bacteria or viruses.

An excellent overview of the defense system is given by Eisen, H.W. (Microbiology, 3rd ed. Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418).

They must be able to attach to endothelial cells so that they can migrate from the circulation to sites of continued inflammation. Furthermore, they must be capable of attaching to antigen-presenting cells so that a normal specific immune response can occur, and finally, they must attach to suitable target cells so that 25 lysis of virus-infected cells or tumor cells can occur.

Recently, leukocyte surface molecules involved in the mediation of such ύ * fortresses were identified using hybridoma technology.

»30 Briefly, monoclonal antibodies directed against human T cells were identified (Davignon, D. et al., Proc.

Natl. Acad. Sci. USA 78: 4535-4539 (1981)) and mouse spleen cells (Springer, T. et al. Eur. J. Immunol. 9: 301-306 - --- - _____i in 2 DK 176020 B1 (1979)), which bound to leukocyte surfaces and inhibited the attachment-related functions described above (Springer, T. et al., Fed. Proc. 44: 2660-2663 (1985)). The molecules identified with these 5 antibodies were called Mac-1 and lymphocyte function-associated antigen-1 (LFA-1). Mac-1 is a heterodimeric island found in macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found in most lymphocytes (Springer, T.A., et al., Immunol. Rev. 68: 111-135 (1982)). These two molecules plus a third molecule, p150.95 (which has a tissue distribution similar to Mac-1), play a role in cellular adhesion (Keizer, G. et al., Eur. J. Immunol.

15: 1142-1147 (1985)).

The above leukocyte molecules have been found to be members of a related family of glycoproteins (Sanchez-Madrid F. et al., J. Exper. Med. 158: 1785-1803 (1983); Keizer, GD et al., Eur. J. Immunol.

15: 1142-1147 (1985)). This glycoprotein family consists of 20 heterodimers with one α chain and one β chain. Although in each of the α-chain of the antigens was different from each other, the β-chain was found to be highly conserved (Sanchez-Madrid, F. et al., J. Exper. Med.

158: 1785-1803 (1983)). The β-chain of the glycoprotein family (sometimes referred to as "CD18") was found to have a molecular weight of 95 kd, whereas the molecular weight of the α chains ranged from 150 kd to 180 kd (Springer, T., Fed.

Proc. 44: 2660-2663 (1985)). Although the α-subunits of the membrane proteins do not have the same extensive homology 4i 30 as β-subunits, closer analysis of the α-subunits of the glycoprotins showed that there are significant similarities between them. Studies on the similarities between the α and β subunits of the LFA-1-related glycoproteins in _______________________________ ____________ are disclosed by Sanchez-Madrid, F. et al. (J. Exper.

With. 158: 586-602 (1983); J ^. Exper. With. 158: 1785-1803 j

(1983)). IN

A group of individuals have been identified who are unable to express normal amounts of any member of this adhesive protein family on * their leukocyte cell surface (Anderson, D.C., et al.,

Fat. Proc. 44: 2671-2677 (1985); Anderson, D.C., et al., J. Infect. Haze. 152: 668-689 (1985)). Lymphocytes from 10 of these patients showed in vitro defects similar to normal counterparts whose LFA-1 family of molecules had been antagonized by antibodies. These individuals were further unable to obtain a normal immune response due to their cells' inability to adhere to cell substrates (Anderson, DC, et al., Fred. Proc. £ 4: 2671-2677 (1985); Anderson , DC, et al., J. Infect. Dis. 152: 668-689 (1985)). These data show that immune responses are attenuated when lymphocytes are unable to attach normally due to the lack of functional adhesion molecules of

LFA-l family. IN

Thus, the ability of lymphocytes to maintain the health and viability of an animal requires that the lymphocytes be able to adhere to other cells (such as endothelial cells). This adhesion has been shown to require cell-cell contacts involving specific receptor molecules found on the cell surface of the lymphocytes. These receptors allow a lymphocyte to attach to other lymphocytes or to. 30 endothelial cells, and other non-vascular cells. The cell surface receptor molecules have been found to be highly related to each other. Persons whose lymphocytes lack these cell surface receptor molecules, j II ----- --- - - - ...... - in 4 DK 176020 B1 exhibits chronic and recurrent infections and other clinical symptoms, including defective antibody reactions.

Since lymphocyte adhesion is involved in the process 5 through which foreign tissue is identified and rejected, an understanding of this process is of significant value in organ transplantation, tissue transplantation, allergy, and oncology.

The present invention relates to monoclonal antibodies and fragments of antibodies capable of inhibiting the function of ICAM-1 and other inhibitors of ICAM-1 function. The invention further relates to diagnostic methods for all the above molecules, as well as pharmaceutical compositions containing the antibodies or fragments thereof, and their use in the preparation of pharmaceutical compositions.

More particularly, the invention relates to a monoclonal antibody, R6-5-D6, capable of binding to a molecule selected from ICAM-1 and functional derivatives of ICAM-1 or molecules capable of binding to a receptor. , found on the surface of a lymphocyte. The invention also encompasses a hybridoma cell capable of producing such an antibody.

The invention further comprises fragments of the antibody capable of binding to the molecule, and further the antibody and fragments thereof in labeled form.

The invention also relates to a method for diagnosing and locating an ICAM-1 expressing tumor cell in a mammal or human, which is believed to contain such a cell, which method comprises: (a) administering an agent containing an detectably labeled binding ligand capable of binding to ICAM-1 to the subject, the ligand being selected from an antibody and a fragment of an antibody, wherein the fragment is capable of binding to ICAM-1, and (b) detection of the binding ligand.

Further, the invention relates to a pharmaceutical composition containing an antibody or toxin-derived antibody capable of binding to a molecule selected from ICAM-1 and a functional derivative of ICAM-1, or a fragment of an antibody or toxin-derived fragment of an antibody, the fragment being capable of binding to a molecule selected from ICAM-1 and a functional derivative of ICAM-1, together with an inert pharmaceutical carrier.

Further, the invention relates to the use of the antibody or fragment thereof capable of binding to ICAM-1 and a functional derivative of ICAM-1 for the preparation of a pharmaceutical composition for the treatment of inflammation arising from a reaction of the specific defense system of a mammal or human, or to suppress the growth of an ICAM-1-expressing tumor or an LFA-1-expressing tumor.

25 In the drawing: FIG. 1, in diagram form, the adhesion between a normal and an LFA-1 defective cell.

FIG. 2 shows, in diagram form, the normal / normal cell adhesion process.

FIG. 3 shows the kinetics of cellular aggregation x in the absence (X) or presence of 50 ng / ml PMA (0).

FIG. Figure 4 shows coaggregation between LFA-1 * and LFA-1 + cells. Carboxyfluorescein diacetate-labeled EBV-transformed cells (104), as indicated in the figure, were mixed with 105 unlabeled autologous cells (black bars) or JY cells (unstained bars) in the presence of PMA.

After 1.5 hours, the labeled cells were counted, in aggregates or free, using the quantitative sample of Example 2. The percentage of labeled cells in aggregates is shown. A representative experiment out of two is shown.

FIG. Figure 5 shows the immunoprecipitation of ICAM-11 and LFA-1 from JY cells. Triton X-100 lysates from JY cells (lanes 1 and 2) or control lysis buffer (lanes 3 and 4) immunoprecipitated with antibody capable of binding to ICAM-1 (lanes 1 and 3) or antibodies which is capable of binding to LFA-1 (lanes 2 and 15 4). Panel A shows results under reducing conditions: Panel B shows results obtained under non-reducing conditions. Molecular weight standards were determined in lane S.

FIG. Figure 6 shows the kinetics of IL-1 and γ-interferon-20 effects on ICAM-1 expression on human dermal fibroblasts. Human dermal fibroblasts were grown to a density of 8 x 10 4 cells / 0.32 cm 2 / well. IL-1 (10 U / ml, black circles) or recombinant γ-interferon (10 U / ml, colored squares) was added and indicated. At 25 minutes, the sample was cooled to 4 ° C and an indirect bond test was conducted. The standard deviation 1 did not exceed 10 percent.

FIG. Figure 7 shows the concentration dependence of IL-1 and γ interferon effects on ICAM-1. Human, then fibroblasts were grown to a density of 8 x 10 4 cells / 0.32 cm 2 / well. IL-2 (unstained circles), recombinant human IL-1 (unstained squares), recombinant mouse IL-1 (black squares), recombinant human γ-interferon (black circles), and recombinant β-interferon DK 176020 B1 ij 7 (unstained) triangles) were added at the indicated dilution and incubated for 4 hours (IL-1) or 16 hours (β- and γ-interferon). The results shown are averages from quadruplicate determinations; the standard deviation 5 did not exceed 10%.

FIG. Figure 8 shows the nucleotide and amino acid sequence of ICAM-1 cDNA. The first ATG is at position 58. Translated sequences corresponding to ICAM-1 tryptic peptides are underlined. The hydrophobic putative signal peptide and transmembrane sequences have a strong underline. N-linked glycosylation sites are framed. The polyadenylation signal AATAAA at position 2976 is indicated by a line above the signal. The sequence shown is for the HL-60 cDNA clone. The endothelial cell-15 cDNA was sequenced over most of its length and showed only minor differences.

FIG. 9 shows the ICAM-1 homologous domains and connection to the immunoglobulin supergenic family.

(A) Arrangement of 5 homologous domains (Dl-5). Two or more 20 identical leftovers that are set up are framed. Residues retained two or more times in NCAM domains as well as residues retained in domains of sets C2 and Cl were aligned with the ICMA-1 internal repeat units. The localization of the predicted β-strands in the ICAM-1 domain is marked with strong dashes and lowercase letters above! the arrays and the known localization of β-strands in immunoglobulin C domains are marked with strong dashes and capital letters below the array. j

The position of the putative disulfide bridge within ICAM-l-j domains is indicated by S-S. (B-D) addition of protein domains homologous to ICAM-1 domains; Proteins were initially prepared by searching NBRF databases using the FASTP program. The protein sequences are MAG, NACM, T cell receptor α subunit V domain,

IgMy chain and α-1-B glycoprotein.

Pig. 10 is a schematic comparison between the secondary structures of ICAM-1 and MAG.

FIG. 1i shows LFA-1 positive EBV-transformed B lymphoblastoid cells binding to ICAM-1 in planar membranes.

FIG. Figure 12 shows LFA-1 positive T lymphoblasts and T lymphoma cells binding to ICAM-1 in plastic bound 10 vesicles.

FIG. Figure 13 shows the inhibition of binding of JY B lymphoblastoid cell binding to ICAM-1 in plastic-bound vesicles by pretreating cells or vesicles with monoclonal antibodies.

FIG. 14 shows the effect of temperature on binding of T lymphoblasts to ICAM-1 in plastic-bound vesicles.

FIG. Figure 15 shows the necessity of divalent cations for binding T-lymphoblasts to ICAM-1 in 20 plastic-bound vesicles.

FIG. Figure 16 shows the effect of anti-adhesion antibodies on peripheral mononuclear blood cell's ability to proliferate in response to recognition of the T cell-associated antigen OKT3. "OK3" indicates the antigen addition 25.

FIG. 17 shows the effect of anti-adhesion antibodies on peripheral mononuclear blood cell's proliferation response in response to the recognition of the nonspecific T cell mitogen, concanavalin A. "CONA" 30 indicates the addition of concanavalin A.

FIG. Figure 18 shows the effect of anti-adhesion antibodies on peripheral mononuclear blood cell proliferation response in response to the recognition of the "keyhole DK 176020 B1 in 9 limpet" hemocyanin antigen. The addition of "keyhole limpet" hemocyanin to the cells is indicated by "KLH".

FIG. 19 shows the effect of anti-adhesion antibodies on peripheral mononuclear blood cells' ability to proliferate in response to the recognition of the tetanus toxoid antigen. The addition of tetanus toxoid antigen to the cells is indicated by "AGN".

FIG. Figure 20 shows the expression of ICAM-1 on human peripheral monocytes. (A) non-cultured responder cells; (B) non-cultured responder X stimulator cells; (C) re sponder cells cultured for 24 h: (D) responder X stimulator cells cultured for 24 h.

(Text: - - - no monoclonal antibody; .......

Mouse immunoglobulin; _ Mouse Anti-ICAM-1).

15 Molecules such as those in the LFA-1 family involved in the cellular adhesion process are referred to as "adhesion molecules".

The present natural binding ligand is referred to as "intercellular adhesion molecule-1" or "ICAM-1".

ICAM-1 is a 76-97 Kd glycoprotein. ICAM-1 is not a heterodimer. A "functional derivative of" ICAM-1 is a compound that has a biological activity (either functional or structural) that is substantially equal to a biological activity of ICAM-1. The term "functional derivatives" is intended to include "fragments", "variants", "analogs" or "chemical derivatives" of a molecule. A "fragment" of a molecule such as ICAM-1 is intended to designate any polypeptide of the molecule. A "variant" of a molecule such as ICAM-1 is intended to denote a molecule having substantially the same structure and function as either the entire molecule or a fragment thereof. One molecule is said to be "substantially equal to" another molecule if both molecules have substantially the same structures or if both molecules have approximately the same biological activity. Thus, provided that two molecules have approximately the same activity, they are considered as variants such as the term used herein, even if the structure of one of the molecules is not found in the other or if the amino acid sequence residues are not identical. An "analog" of a molecule, such as ICAM-1, is intended to denote a molecule substantially similar in function either to the entire molecule or to a fragment thereof. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties which are not normally part of the molecule. Such 15 moieties may improve the solubility, absorption and biological half-life of the molecule, etc. The moiety may alternatively reduce the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. moieties capable of mediating such effects are specified in Reminton's Pharmaceutical Sciences (1980). "Toxin-derived" molecules form a special class of "chemical derivatives". A "toxin-derived" molecule is a molecule (such as ICAM-i or an antibody) that contains a toxin moiety. The binding of such a molecule to a cell brings the toxin moiety in close proximity to the cell, thereby promoting cell death.

Any suitable toxin moiety may be used; however, it is preferred to use toxins such as castor toxin, dieththeria toxin, radioisotopic toxins; membrane-channel-forming toxins, etc. Methods for coupling such moieties to a molecule are well known in the art.

An antigenic molecule such as ICAM-1 or members of the LFA-1 family of molecules is naturally expressed on the surface of lymphocytes. Thus, the introduction of such cells into suitable animals, as by intraperitoneal injection, etc., will result in the production of antibodies capable of binding to ICAM-1 or members of the LFA-1 family of molecules. The serum from such an animal can, if desired, be removed and used as a source of polyclonal antibodies capable of binding to these molecules. However, it is preferred to remove splenocytes from such animals, to fuse such spleen cells with a myeloma cell line and allow such fusion cells to form a hybridoma cell that secretes monoclonal antibodies capable of binding to ICAM-1 or members of LFA. 1 the family of molecules.

The hybridoma cells obtained in the same manner as described above can be screened by a variety of methods for identifying desired hybridoma cells that secrete antibody capable of binding either to ICAM-1 or to members of LFA-1. 20 the family of molecules. In a preferred screening assay, such molecules are identified by their ability to inhibit the aggregation of Epstein-Barr virus-transformed cells. Antibodies capable of inhibiting such aggregations are then subjected to further screening to determine whether they inhibit such aggregation by binding to ICAM-1, or by binding to a member of the LFA-1 family of molecules. Any agent capable of distinguishing ICAM-1 from the LFA-1 family of molecules can be used in such a screening. Thus, the antigen bound by the antibody can e.g. assayed by immunoprecipitation and polyacrylamide gel electrophoresis. Thus, if the bound antigen is a member of the LFA-1 family of molecules, the immunoprecipitated antigen will be a dimer, whereas a single molecular weight portion would have been immunoprecipitated if the bound antigen is ICAM-i. Furthermore, it is possible to distinguish between these antibodies that bind to members of the LFA-1 family of molecules from those that bind to ICAM-1 by screening for the antibody's ability to bind to cells, such as granulocytes, which express LFA-1 but not ICAM-1. The ability of an antibody (known to inhibit cellular aggregation) to bind to granulocytes indicates that the antibody is capable of binding to LFA-1. The absence of such binding indicates that an antibody is capable of recognizing ICAM-1. The ability of an antibody to bind to a cell, such as a granulocyte, can be demonstrated by methods usually employed by one of ordinary skill in the art. Such methods include immunoassays, cellular agglutination, filter binding studies, antibody precipitation, etc.

Alternatively, the aforementioned anti-aggregation antibodies can be identified by measuring their ability to differentially bind to cells expressing ICAM-1 (such as activated endothelial cells) and their inability to bind to cells that do not express ICAM-1. As will be appreciated by those skilled in the art, the above tests may be modified or performed in a different order to obtain a series of potential screening samples, each capable of identifying and distinguishing antibodies capable of binding to ICMA-1 versus members of the LFA-1 family of molecules.

The present anti-inflammatory agents may be obtained by natural (such as by causing an animal, plant, fungus, bacterium, etc. to produce a non-immunoglobulin antagonist of ICAM-1, or by obtaining a 13 DK 176020 B1 animals for producing polyclonal antibodies capable of binding to ICAM-1); by synthetic methods (such as using Merrifield's method of synthesizing polypeptides to synthesize ICAM-1, functional derivatives of ICAM-1, or protein antagonists of ICAM-1 (either immunoglobulin or non-immunoglobulin)); by hybridoma technology (such as the preparation of monoclonal antibodies capable of binding to ICAM-1); or by recombinant technology (such as preparing the subject anti-inflammatory agents in various hosts (i.e., yeast, bacteria, fungi, cultured mammalian cells, etc.), or from recombinant plasmids or virus vectors).

The choice of method to be used will depend on factors such as utility, desired yield, etc. It is not necessary to use only one of the above methods, methods or technologies to produce a particular anti-inflammatory agent; the above methods, methods and technologies 20 can be combined to obtain a particular anti-inflammatory agent.

A. Identification of the LFA-1 Binding Partner (ICAM-1).

25 1. Tests for LFA-1-dependent aggregation.

Many Epstein-Barr virus-transformed cells exhibit aggregation. This aggregation can be increased in the presence of phorbol esters. Such homotypic aggregation (i.e., aggregation involving only one cell type) has been shown to be blocked by anti-LFA-1 antibodies (Rothlein, R. et al., J. Exper. Med. 163: 1132 -1149 (1986)), the reference of which is incorporated herein by reference). Thus, the extent of LFA-1-dependent binding can be determined by testing the extent of spontaneous or phorbol ester-dependent aggregate formation.

An agent that interferes with LFA-1-dependent aggregation can be identified by the use of a sample capable of determining whether an agent interferes with the spontaneous or phobolsteroid-dependent aggregation of Epstein-Barr virus-10 transformed cells. Most Epstein-Barr virus-transformed cells can be used in such a sample as long as the cells are capable of expressing the LFA-1 receptor molecule. Such cells can be prepared according to the technique disclosed by Springer, 15 T.A. et al., J. Exper. With. 160: 1901-1918 (1984), which reference is incorporated herein by reference thereto. Although any such cell can be used in the LFA-1 dependent binding assay of the invention, it is preferred to use cells from the JY cell line 20 (Terhost, CT et al., Proc. Natl. Acad. Scl. USA 22: 910 (1976)) . The cells can be grown in any suitable culture medium; however, it is preferred to culture the cells in RMPI 1640 culture medium supplemented with 10% fetal calf serum and 50 µg / ml gentamycin (Gibco Laboratories, NY). The cells should be grown under conditions suitable for mammalian cell proliferation (i.e., at a temperature of usually 37 ° C and in an atmosphere of 5% CO 2, at a relative humidity of 95%, etc.).

15 DK 176020 B1 2. LFA-1 binding to ICAM-1.

Humans have been found whose lymphocytes lack the family of LFA-1 receptor molecules (Anderson, D.C.

5 et al., Fed. Proc. 44: 2671-2677 (1985); Anderson, D.C. et al., J. Infect. Haze. 152: 668-689 (1985)). Such people are said to suffer from leukocyte adhesion deficiency (LAD). EBV-transformed cells from such humans are assembled neither spontaneously nor in the presence of phorbol esters in the above aggregation assay. When such cells are mixed with LFA-1-expressing cells, aggregation is observed (Rothlein, R. et al., J. Exper.

With. 163: 1132-1149 (1986)) (Fig. 1). It is of great importance that these aggregates do not form if these 15 cells are incubated in the presence of anti-LFA-1 antibodies. Although aggregation required LFA-1 indicated the ability of LFA-1 deficient cells to form aggregates with LFA-1-containing cells such that the LFA-1 binding partner was not LFA-1, but rather a novel cell adhesion molecule. FIG. Figure 1 shows the cellular adhesion mechanism.

B. Intercellular Adhesion Molecule-1 (ICAM-1).

25

The subject intercellular adhesion molecule ICAM-1 was first identified and partially described according to the method of Rothlein, R. et al. (J. Immunol. 137: 1270-1274 (1986)). For detection of 30 of the ICAM-1 molecule, monoclonal antibodies were prepared from mouse spleen cells immunized with cells in humans with genetic defect in LFA-1 expression.

The antibodies obtained were screened for their ability to inhibit the aggregation of LFA-1-expressing cells (Fig. 2). Specifically, 5 mice were immunized with EBV-transformed B cells from LAD patients who do not express the LFA-1 antigen. Spleen cells from these animals were then removed, fused with myeloma cells and allowed to become monoclonal antibody-producing hybridoma cells. Normal human EBV-transformed ΒΙΟ cells expressing LFA-1 were then incubated in the presence of the hybridoma cell monoclonal antibody to identify any monoclonal antibody capable of inhibiting the phorbol ester-mediated, LFA-1 dependent in 15 spontaneous aggregation of the EBV-transformed B cells.

Because the hybridoma cells were derived from cells that had never encountered the LFA lanthigen, no monoclonal antibody to LFA-1 was produced. Thus, any antibody found to inhibit aggregation must be capable of binding to an antigen that, although not LFA-1, participates in the LFA-1 adhesion process. Although any method for obtaining such monoclonal antibodies can be used, it is preferred to obtain ICAM-1 binding monoclonal antibodies by immunization of BALB / C mice, methods and plans described by Rothlein, R. et al., (J. Immunol. 137: 1270-1274 (1986)) is used with Epstein-Barr virus-transformed human peripheral blood mononuclear cells with an LFA-1 defect. Such cells are disclosed by Springer, 30 T.A., et al., (J. Exper. Med. 160; 1901-1918 (1984)).

In a method for producing and detecting antibodies capable of binding to ICAM-1, mice are immunized with either EBV-transformed B-! B1 cells expressing both ICAM-1 and LFA-1 or more, preferably with TNF-activated endothelial cells expressing ICAM-1 but not LFA-1. In the most preferred method of producing hybridoma cells producing anti-ICAM-1 antibodies, a Balb / C mouse was sequenced with JY cells and differentiated.

tiered U937 cells (ATCC CRL1593). The spleen cells from such animals were removed, fused with myeloma cell line. clay and allowed to develop in antibody-producing I 10 hybridoma cells. The antibodies are screened for their ability to inhibit the LFA-1-dependent, phorbol ester-induced aggregation of an EBV-trans-β proliferated cell line, such as JY cells expressing both the LFA-1 receptor and ICAM-1. As shown by Rothlein, I 15 R., et al. (J. Immunol. 137; 1270-274 (1987)), antibodies capable of inhibiting such aggregation are then tested for their ability to inhibit the phorbol ester-induced aggregation of a cell line such as SKW3 (Dustin, M., et al., J. Exper. Med.

20 165: 672-692 (1987)) whose ability to spontaneously aggregate in the presence of a phorbol ester is inhibited by antibody capable of binding LFA-1 but is not inhibited by anti-ICAM-1 antibodies. Antibodies capable of inhibiting the phorbol ester-induced aggregation of cells, such as JY cells, but unable to inhibit the phorbol ester-induced aggregation of cells such as SKW3 cells are likely to be anti-ICAM-1 antibodies. Alternatively, antibodies capable of binding to ICAM-1 can be identified by screening for antibodies capable of inhibiting the LFA-1-dependent aggregation of LFA expression cells (such as JY cells), but which are unable to bind to cells expressing LFA-1 but little DK 176020 B1

18 I

or no ICAM-1 (such as normal granulocytes) or capable of binding to cells expressing ICAM-1 but not LFA-1 (such as TNF-activated endothelial cells). Another alternative is immunoprecipitation 5 from cells expressing ICAM-1, LFA-1, or both, using antibodies that inhibit the LFA-1-dependent aggregation of cells, such as JY cells, and determine some molecular properties of the molecule that precipitated with the antibody through SDS-PAGE or a 10 equivalent method. If the properties are the same as for ICAM-1, the antibody is presumed to be an anti-ICAM-1 antibody.

Using monoclonal antibodies prepared analogously to the above, the ICAM-15 1 cell surface molecule was purified and its properties determined. ICAM-1 was purified from human cells or tissues using monoclonal antibody affinity chromatography, in such a method coupling a monoclonal antibody reactive with ICAM-1 to an inert column matrix. Any method of obtaining such a coupling may be employed; however, it is preferred to use the method described by Oettgen, H.C. et al., J. Biol. Chem. 259: 12034 (1984)). When a cell lysate passes through the matrix, it is adsorbed into the existing ICAM-1 molecule and retained by the matrix. by changing the pH or ion concentration in the column, the bound ICAM-1 molecules can be eluted from the column. Although any suitable matrix can be used, it is preferred to use sepharose (Pharmacia) as the matrix material. The preparation of column matrices and their use in protein purification is well known in the art.

In a manner understood by one of ordinary skill in the art, the above tests can be used to identify compounds which are capable of attenuating or inhibiting the degree! or the extent of cellular adhesion.

ICAM-1 is a cell surface glycoprotein expressed on non-hematopoietic cells, such as vascular endothelial cells, thymus epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte, B cells and dendrite cells in tonsils, lymph nodes and "Peyer's patches".

10 ICAM-1 is strongly expressed on vascular endothelial cells in T-cell regions of lymph nodes and tonsils, exhibiting reactive hyperplasia. ICAM-1 is expressed in small amounts on peripheral blood lymphocytes. Phorbol ester-stimulated differentiation of some myelomonocytic cell lines greatly increases ICAM-1 expression. Thus, ICAM-1 is preferably expressed at sites of inflammation and is generally not expressed by passive cells. ICAM-1 expression on dermal fibroblasts is increased 3–5-fold with either interleukin 1 or γ-interferon at 20 concentrations of 10 U / ml over 4 or 10 hours, respectively. The induction is dependent on protein and mRNA synthesis and is reversible.

ICAM-1 exhibits molecular weight uniformity in different cell types with a molecular weight of 97 kd in fibroblasts, 114 kd in the myelomonocytic cell line U937 and 90 kd in the B lymphoblastoid cell JY. ICAM-I biosynthesis has been shown to involve an approx. 73 kd intercellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment (which inhibits glycosylation) 30 has a molecular weight of 55 kd.

ICAM-1 isolated from phorbol ester stimulated U937 cells or from fibroblast cells provides a similarly large product with a molecular weight of 60 kd after chemical deglycosylation. ICAM-1 monoclonal antibodies interfere with the adhesion of phytohaemaglutinin blasts to LFA-1 deficient cell lines. Pre-treatment of fibroblasts, but not lymphocytes, with 5 monoclonal antibodies capable of binding ICAM-1 inhibits lymphocyte fibroblast adhesion. Pre-treatment of lymphocytes, but not fibroblasts, with! In addition, antibodies against LFA-1 have been shown to inhibit lymphocyte fibroblast adhesion.

Thus, ICAM-1 is the CD 18 complex's binding ligand on leukocytes. It is inducible on fibroblasts and endothelial cells in vitro by inflammatory mediators such as IL-1, γ-interferon and tumor necrosis factor within a time frame consistent with lymphocyte infiltration in inflammatory lesions in vivo (Dustin, ML, et al J. Immunol 137: 245-254, (1986); Prober, JS, et al., J ♦ Immunol 137: 1893-1896, (1986)). ICMA-1 is further expressed on non-hematopoietic cells, such as vascular endothelial cells, thymus epithelial cells, other epithelial cells, and fibroblasts and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center B cells and dendritic cells. , lymph nodes and "Peyer's patches" (Dustin, ML et.

25, Immunol 137: 245-254, (1986)). ICAM-1 is expressed on keratinocytes in benign inflammatory lesions such as allergic eczema, lichen ruber, exanthema, urticaria and blistering diseases. Allergic skin reactions, provoked by the application of a hapten to the skin 30 against which the patient is allergic, also showed potent ICAM-1 expression on keratinocytes. On the other hand, toxic patches on the skin did not show icam-1 expression on the keratinocytes. ICAM-1 is found on keratinocytes from biopsies of skin lesions from various dermatological diseases and ICAM-1 expression is induced on damage from allergic lap tests, whereas keratinocytes from toxic lap test injuries did not express ICAM-1.

Therefore, ICAM-1 is a cell substrate to which lymphocytes can be attached so that the lymphocytes can! migrate to sites of inflammation and / or carry out various effector function clones that contribute 10 to this inflammation. Such functions include the production of antibody, lysis of virus-infected target cells, etc. The term "inflammation" as used herein is intended to include only responses from the specific defense system. As used herein, the term "specific defense system" is intended to refer to the component of the immune system that reacts in the presence of specific antigens. Inflammation is said to occur as a reaction from the specific defense system if the inflammation 20 is caused by, mediated by, or linked to a reaction from the specific defense system. Examples of inflammation arising from a reaction of the specific defense system include the response to antigens such as rubella virus, autoimmune diseases, delayed type of hypersensitivity reaction mediated by T cells (as seen, for example, in humans whose test is "positive" in the Mantaux test), etc.

22 DK 176020 B1 C. Cloning of the ICAM-1 gene.

Any of a variety of methods can be used for cloning to the ICAM-1 gene. Such a method involves the analysis of a shuttle vector library of cDNA inserts (derived from an ICAM-1-expressing cell) for the presence of an insert containing the ICAM-1 gene. Such analysis can be performed by transfecting cells with the vector and then testing for ICAM-1 expression. The preferred method of cloning this gene involves determining the amino acid sequence of the ICAM-1 molecule. To accomplish this task, ICAM-1 protein must be purified and analyzed by automated sequencers. Alternatively, the molecule may be subjected to fragmentation with cyanogen bromide or with proteases such as papain, chymotrypsin or trypsin (Oike, Y. et al., J. Biol. Chem. 257: 9751-9758 (1982); Liu, C. et al. ., Int. J. Pept. Protein

Res. 21: 209-215 (1983)).

Although it is possible to determine the entire amino acid sequence of ICAM-1, it is preferred to determine the peptide fragment sequence of the molecule. If the peptide is more than 10 amino acids long, the sequence information is usually sufficient to clone a gene such as the ICAM-1 gene.

The sequence of amino acid residues in a peptide is denoted herein either by the use of their commonly used 3-letter designations or by their single-letter designations. A list of these 3-book 30-letter and 1-letter designations can be found in text books such as Biochemistry, Lehninger, A., Worth Publishers, New York, NY (1970). When such a sequence is erected vertically, it is intended that the amino-terminal residue should be at the top of the list and the carboxy-terminal residue of the peptide should be at the bottom of the list. Similarly, when the sequence is erected horizontally, it is intended that the amino-terminal residue be left, whereas the 5-carboxy-terminal residue should be to the right. The amino acid residues in a peptide can be separated by hyphens.

Such hyphens are intended solely to facilitate a sequence presentation. As a purely illustrative example, the amino acid sequence designated: 10 -Gly-Ala-Ser-Phe- indicates that an Ala residue is bound to Gly's carboxy group and that in a Ser residue is bound to the carboxy group of Ala residue and 15 to a Phe- residue amino group. The designation further indicates that the amino acid sequence contains tetrapeptide Gly-Ala-Ser-Phe. The term is not intended to limit the amino acid sequence of this one tetrapeptide, but it is intended to include (1) the tetrapeptide containing one or more amino acid residues bound to either its amino or carboxy terminus, (2) the tetrapeptide containing a or more amino acid residues linked to both its amino and carboxy ends, (3) the tetrapeptide, which contains no further amino acid residues.

Once one or more suitable peptide fragments have been sequenced, DNA sequences capable of encoding them are examined. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid (Watson, J.D., I: Molecular Biology of the Gene, 3rd ed., W.A. Benjamin,

Inc., Menlo Park, CA (1977), pp. 356-357). The peptide fragments are analyzed to identify sequences of amino acids which can be encoded by oligonucleotides with the lowest degree of degeneration. This is preferably accomplished by identifying sequences containing amino acids encoded by only a single codon.

Although such amino acid sequences may occasionally be encoded by only a single oligonucleotide, the amino acid sequence can often be encoded by any nucleotide from a set of similar oligonucleotides. While all members of the kit contain oligonucleotides which are capable of encoding the peptide fragment, and thus potentially contain the same nucleotide sequence as the gene encoding the peptide fragment, it is essential that only one member of the kit contains a nucleotide sequence that is identical with this nucleotide sequence of this gene. Because this member exists in the kit and is capable of hybridizing to DNA, even in the presence of other members of the kit, it is possible to use the unfractionated set of oligonucleotides in the same way as a single oligonucleotide would be used for cloning the gene , which encodes the peptide.

In a manner analogous to the above, one may employ an oligonucleotide (or set of oligonucleotides) containing a nucleotide sequence complementary to the oligonucleotide sequence or set of sequences capable of encoding the peptide fragment.

A suitable oligonucleotide or set of oligonucleotides capable of coding for a fragment of the ICAM-1 gene (or complementary to such an oligonucleotide, or set of oligonucleotides) is identified (using the above method), synthesized and synthesized. hybridisers, by methods well known in the art, to a DNA or, more preferably, a cDNA preparation derived from human cells capable of expressing ICAM-1 gene sequences. Methods for nucleic acid hybridization are disclosed by Maniatis, T. et al., In: Molecular Cloning, 5a Laboratory Manual, Coldspring Harbor, NY (1982), and by Haymes, B.D. et al., In: Nucleic Acid Hybrizatlon, A Practical Approach, IRL Press, Washington, DC (1985), which references are incorporated herein by reference.

The DNA or cDNA source used will preferably have been enriched for ICAM-1 sequences. Such an enrichment can very easily be obtained from cDNA obtained by extracting RNA from cells grown under conditions which induce ICAM-1 synthesis (such as U937 grown in the presence of phorbol esters, etc.).

Techniques such as, or similar to those described above, have successfully enabled cloning of genes for human aldehyde dehydrogenases (Hsu, L.C.

et al., Proc. Natl. Acad. Sci. USA 82: 3771-3775 (1985)), fibronectin (Suzuki, S. et al., Eur. Mol.

Biol. Organ. J. £: 2519-2524 (1985)), the human estrogen receptor gene (Walter, P. et al., Proc, Natl.

Acad. Sci. USA 82: 7889-7893 (1985)), tissue type plasminogen activator (Pennica, D. et al. Nature 301: 214-221 (1983)) and human end placental basic phosphatase complementary DNA (Kam, W. et al., Proc. Natl. Acad.

Sci. USA 82: 8715-8719 (1985)).

In a preferred alternative method for cloning the ICAM-1 gene, a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA, from a cell capable of expressing ICAM-1 into an expression vector. The library is then screened for members capable of expressing a protein that binds to antibody that has a nucleotide sequence capable of encoding polypeptides which have the same amino acid sequence as ICAM-1 or fragments of ICAM-1.

The cloned ICAM-1 gene, obtained by the above methods, can be operably bound to an expression vector and introduced into bacterial or eukaryotic cells to produce ICAM-1 protein.

Techniques for such manipulations are indicated by 10 Maniatis. T. et al., Supra, and are well known in the art.

D. Uses of LFA-1 dependent aggregation samples. r 15

The above test, where one is able to measure LFA-1-dependent aggregation, can be used to identify drugs that act as antagonists to inhibit the extent of LFA-1-dependent aggregation.

Twenty such antagonists may act by impairing the ability of LFA-1 or ICAM-11 to mediate aggregation. Thus, such agents include immunoglobulins, such as an antibody capable of binding to either LFA-1 or ICAM-1. Non-immunoglobulin (i.e., chemical) agents 25 can be further tested using the above test to determine if they are antagonists of LFA-1 aggregation.

27 USE 176020 B1 E. Uses of antibodies capable of binding to ICAM-1 receptor proteins.

I. Anti-inflammatory agents.

5

Monoclonal antibodies to members of the CD 18 complex inhibit the adhesion-dependent functions of many leukocytes, including binding to endothelium (Haskard, D., et al., J ♦ Immunol. 137; 2901-2906 (1986)), homotypic adhesions (Rothlein, R. , et al ♦, J. Exp. Med. 163: 1132-1149 (1986)), antigen- and mitogen-induced proliferation of lymphocytes (Davignon, D., et al., Proc. Natl. Acad. Sci., USA 78: 4535-4539 (1981)), antibody formation (Fischer, A., et al., J.

Immunol. 136: 3198-3203 (1986)), and effector functions of all leukocytes, such as the lytic: activity of cytotoxic T cells (Krensky, AM, et al. ♦ J ♦ Immunol. 132: 2180-2182 (1984)), macrophages (Strassman, G. , et al.

J. Immunol. 136: 4328-4333 (1986)), and all cells involved in antibody-dependent cell cytotoxic reactions (Kohi, S., et al., J. Immunol. 133: 2972-2978 (1984)). In all the above functions, the antibodies inhibit the ability of the leukocyte to adhere to the appropriate cell substrate, which in turn inhibits the final result.

As discussed above, the binding of ICAM-1 molecules to members of the LFA-1 family of molecules is of great importance in cellular adhesion. Through the adhesion process, lymphocytes are able to continuously examine an animal for the presence of foreign antigens.

Although these processes are usually desirable, they are also the cause of organ transplant rejection, tissue transplant rejection, and many autoimmune diseases. Thus, any agent capable of attenuating or inhibiting cellular adhesion would be highly desirable in patients undergoing organ transplants, tissue transplants, or in autoimmune patients.

Monoclonal antibodies capable of binding to ICAM-1 are particularly useful as anti-inflammatory agents in a mammal. Such agents differ substantially from conventional anti-inflammatory agents in that they are capable of selective inhibitory adhesion and do not produce other side effects such as nephrotoxicity found with usual agents. Monoclonal antibodies capable of binding to ICAM-1 can therefore be used to prevent organ or tissue rejection, or to modify autoimmune reactions without fear of such side effects in the mammal.

It is of great importance that the use of monoclonal antibodies capable of recognizing ICAM-1 enables organ transplants to be performed even between individuals with high HLA disparity.

29 DK 176020 B1 2. Suppressors of delayed-type hypersensitivity reaction.

Since ICAM-1 molecules are most often expressed at 5 sites of inflammation, such as those involved in delayed-type hypersensitivity reaction, antibodies (especially monoclonal antibodies) capable of binding to ICAM-1 molecules have therapeutic potential. to suppress or eliminate such reactions. This possible therapeutic application can be utilized in two ways. First, an agent containing a monoclonal antibody to ICAM-1 can be administered to a patient suffering from delayed-type hypersensitivity reaction. Such agents may e.g. is given to a subject who has been in contact with antigens such as toxic ephew, toxic oak, etc. In another embodiment, the monoclonal antibody capable of binding to ICAM-1 is administered to a patient along with an antigen. to prevent a subsequent inflammatory reaction. Thus, this co-administration of an antigen and an ICAM-1 binding monoclonal antibody may temporarily cause an individual to subsequently tolerate exposure to this gene.

25 3. Treatment of chronic inflammatory disease.

Since LAD patients lacking LFA-1 do not achieve an inflammatory response, it is believed that antagonism of the natural ligand of LFA-1, ICAM-1, will also inhibit inflammatory response. The ability of antibodies to inhibit inflammation against ICAM-1 provides the basics for their therapeutic use in the treatment of chronic inflammatory and autoimmune diseases such as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis 5 (EAE) multiple forms of sclerosis, diabetes Reynaud's syndrome, rheumatoid arthritis, etc. The monoclonal antibodies capable of binding ICAM-1 can usually be used to treat these diseases currently being treated through steroid therapy.

4. Diagnostic and prognostic uses.

15

Since ICAM-1 is often expressed at sites of inflammation, monoclonal antibodies capable of binding to ICAM-1 can be used as a means of describing or visualizing sites of infection and inflammation in a patient, in such use being labeled the monoclonal antibodies detectable through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.) fluorescent markers, para-magnetic atoms, etc. Methods for carrying out such labeling are well known in the art.

Clinical use of antibodies for diagnostic description is disclosed by Grossman, H.B., Urol. Clin.

North Arner. 13 :: 465-474 (1986)), Unger, E.C. et al ..

Invest. Radiol. 20: 693-700 (1985)), and Khaw, B.A. et al., Science 209: 295-297 (1980)).

The presence of inflammation can also be detected through the use of binding ligands, such as mRNA, cDNA or DNA, which bind to ICAM-1-31 'DK 176020 B1 gene sequences or to ICAM-1 mRNA sequences in cells expressing ICAM-1. Techniques and conducting such hybridization tests are described by Maniatais, T. (supra).

The detection of feet of such detectable labeled antibodies is indicative of a site of inflammation or tumor development. In one embodiment, this study is conducted for inflammation by removing tissue samples or blood and incubating such samples 10 in the presence of detectably labeled antibodies. In a preferred embodiment, this technique is performed in a non-invasive manner through the use of magnetic imaging, fluorography, etc. Such a diagnostic test can be used to monitor organ transplant rejection for early signs of potential tissue rejection. In addition, such tests may be conducted in an attempt to determine an individual's predilection for rheumatoid arthritis or other chronic inflammatory diseases.

5. Supplement to the introduction of antigenic material administered for therapeutic and diagnostic purposes.

25

Immune reactions to therapeutic or diagnostic agents such as bovine insulin, interferon, tissue type palsminogen activator, or murine monoclonal antibodies substantially impair the therapeutic or diagnostic value of such agents and may actually cause diseases such as serum sickness. Such a situation can be remedied by the use of the antibodies in question. In this embodiment, then antibodies forming antibodies will be administered together with a therapeutic or diagnostic agent. The addition of the antibodies prevents the recipient from recognizing the agent and therefore prevents the recipient from initiating immune response to it. Absence of such an immune response allows the patient to receive additional administrations of the therapeutic or diagnostic agent.

in 10 F. Applications of Intercellular Adhesion Molecule-1 (ICAM-1).

ICAM-i is a binding partner of LFA-1. As such, ICAM-1 15 or its functional derivatives can be used interchangeably with antibodies capable of binding to LFA-1 in the treatment of disease. Thus, in solute form, such molecules can be used to inhibit inflammation, organ rejection, tissue rejection, etc. ICAM-1 or its functional derivatives can be used in the same way as anti-ICAM-1 antibodies to decrease the immunogenicity of therapeutic or diagnostic agents.

ICAM-1, its functional derivatives, and its antagonists can be used to block metastasis or proliferation of tumor cells expressing either ICAM-1 or LFA-1 on their surfaces. A wide variety of methods can be used to achieve such a goal. The migration of hematopoietic cells requires e.g. LFA-I-ICAM-1 binding. Therefore, such a binding antagonist suppresses this migration and blocks metastasis of leukocyte-derived tumor cells. Toxin-derivatized molecules that are in 33.

Alternatively, DK 176020 B1 capable of binding to either ICAM-1 or a member of the LFA-1 family of molecules may be administered to a patient. When such toxin-derived molecules bind to tumor cells expressing ICAM-1 or a member of the LFA-1 family of molecules, the presence of the toxin kills the tumor cells, thereby inhibiting the proliferation of the tumor.

^ G. Applications of non-immunoglobulin antagonists of ICAM-1-dependent adhesion.

ICAM-1-dependent adhesion may be inhibited by non-immunoglobulin antagonists capable of binding to ICAM-1 or LFA-1. An example of a non-immunoglobulin antagonist of ICAM-1 is LFA-1. An example of a non-immunoglobulin antagonist that binds to LFA-1 is ICAM-1. Through the use of the above tests, non-immunoglobulin antagonists 20 can be further identified and purified. Non-immunoglobulin antagonists of ICAM-1-dependent adhesion can be used for the same purpose as antibodies to LFA-1 or antibodies to ICAM-1.

25 H. Administration of the funds in question.

The therapeutic effects of ICAM-1 can be achieved by providing a patient with the entire ICAM-1 molecule or any therapeutically active peptide fragment thereof.

ICAM-1 and its functional derivatives can be obtained either synthetically, through the use of recombinant DNA technology or by proteolysis. The therapeutic benefits of ICAM-1 can be increased through the use of functional derivatives of ICAM-1, which contain additional amino acid residues, to increase the coupling to carriers or increase the activity of ICAM-1. The invention 5 further encompasses functional derivatives of ICAM-1 which lack certain amino acid residues or which contain altered amino acid residues as long as such derivatives possess the ability of cellular adhesion action.

Both the antibodies in question and said ICAM-1 molecule are said to be "substantially free of natural contamination" if the compositions containing them substantially do not contain materials normally and naturally found in these products.

When giving a patient antibodies, or fragments thereof capable of binding to ICAM-1, or when giving ICAM-1 (or in fragment, variant or derivative thereof) to a receiving patient, the dose of administered agent varies depending on 20 factors such as the patient's age, weight, height, gender, general medical condition, prior medical history, etc. It is usually desirable to provide the recipient with a dose of antibody which is within the range of about 5,000. 1 pg / kg to approx. 10 mg / kg 25 (patient body weight), although a smaller or larger dose can be administered. When administering to a patient, ICAM-1 molecules or their functional derivatives, it is preferred to administer such molecules at a dose which is also within the range of approx. 1 µg / kg to 10 30 mg / kg (patient body weight) although a smaller or larger dose can also be administered.

As discussed below, the therapeutically effective dose may be reduced if anti-ICAM-1 anti-DK 176020 B1. The substance is co-administered with an anti-LFA-1 antibody.

As used herein, two antibodies (or their functional derivatives) are said to be co-administered to a patient if they have been administered to the patient within a period of time so that both compounds can be detected in the patient's serum.

Both the antibody capable of binding to ICAM-1 and ICAM-1 itself can be administered to! patients intravenously, intramuscularly, subcutaneously, internally or parenterally. When antibody or ICAM-1 is administered by injection, administration may be by continuous infusion, or by single or multiple boluses.

It is intended with the present anti-inflammatory agents to provide them to recipient individuals in an amount sufficient to suppress inflammation. An amount is said to be sufficient to "suppress" inflammation if the dose, mode of administration, etc. of the agent is sufficient to attenuate or prevent inflammation.

The present anti-inflammatory agents may be administered either before or upon the onset of inflammation (to suppress the expected inflammation) or after the onset of inflammation.

An agent is said to be "pharmacologically acceptable" if its administration can be tolerated by a receiving patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a receiving patient.

The antibody and ICAM molecules can be formulated according to known methods for the preparation of pharmaceutically useful agents, where these materials or their functional derivatives are combined in a mixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, including, 5 other human proteins, e.g. human serum albumins described e.g. in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). To form a pharmaceutically acceptable agent suitable for effective administration, such agents 10 will contain an effective amount of anti-ICAM-1 antibody or ICAM-1 molecule or their functional derivatives, together with a suitable amount of a carrier vehicle.

Additional pharmaceutical methods can be used to control duration of action. Controlled release preparations can be obtained by the use of polymers for complexing or absorbing anti-ICAM-1 antibody or ICAM-1 or their functional derivatives. The controlled loading can be accomplished by selecting appropriate macromolecules 20 (e.g., polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose or protamine, sulfate) and the concentration of macromolecules and the methods of incorporation to control release. Another suitable method for controlling the duration of action of controlled release preparations is the incorporation of anti-ICAM-1 antibody or ICAM-1 molecules, or their functional derivatives, into particles of a polymeric material such as polyesters, polyamino acids, hybrids. 30 drugs, poly (lactic acid) or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these agents into the polymeric particles, it is possible to enclose these materials in microcapsules manufactured 37 e.g. by coacervation techniques or by interfacial polymerization, e.g. hydroxymethyl cellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules or 1 colloidal drug delivery systems, e.g. Ilposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules or in macroemulsions. Such techniques are described in Reminton's Pharmaceutical Sciences (1980).

The invention is now further illustrated by the following 10 examples.

Example 1

Cultivation of mammalian cells.

The subject EBV transformed and hybridoma cells were usually stored in RMPI 1640 culture medium supplemented with 20 mM L-glutamine, 50 µg / ml gentamicin and 10% fetal bovine (or fetal calf) sera. The cells were grown at 37 ° C in an atmosphere of 5% CO 2, 95% humidity.

To establish Epstein-Barr virus (EBV) transformants, 106 T-cell poor peripheral blood mononuclear cells / ml were incubated in RPMI 1640 medium supplemented with 20% fetal calf serum (PCS) and 50 µg / ml gentamicin for 16 hours. with EBV-containing supernatant of 25 B95-8 cells (Thorley-Lawson, DA et al., J. Exper.

With. 146: 495 (1977)). Cells in 0.2 ml aliquot were placed in 10 microtiter wells. The medium was replaced with RPMI 1640 medium (supplemented with 20% fetal calf serum and 50 µg / ml gentamicin) until cell growth was detected.

The 30 cells grew in most wells and expanded in the same medium. Phytohaemagglutinin (PHA) blasts were established at 106 cells / ml in RPMI 1640 medium (supplemented with 20% fetal calf serum) containing a 38 dilution of PHA-P (Difco Laboratories, Inc., Detroit, MI). PHA lines were expanded with interleukin 2 (IL-2) conditioned medium and pulsed weekly with pha (Cantrell, D.A. et al., J. Exper. Med.

158: 1895 (1983)). The above method is described by Springer, T. et al., J. Exper. Med ♦ 160: 1901-1918 (1984). Cells obtained by the above method are then screened with anti-LFA-1 antibodies to determine whether they express the LFA-1 antigen.

Ten such antibodies are disclosed by Sanches-Madrid, F. et al., J. Exper. With. 158: 1785 (1983).

Example 2.

Tests for cellular aggregation and adhesion.

To test the extent of cellular adhesion, aggregation samples were used. The cell lines used in such samples were washed twice with RPMI 1640 medium containing 5 mM Hepes buffer (Sigma Chemical Co., St. Louis) and resuspended to a concentration of 2 x 10 6 cells / ml. To flat-bottomed, 96-well microtiter plates (no. 3596; Costar, Cambridge, MA) were added 50 μΐ of appropriate monoclonal antibody supernatant or 50 μl complete medium with or without purified monoclonal antibodies, 50 μΐ complete medium containing 200 ng / ml. ml of the phorbol ester, phorbol myristate acetate (PMA) and 100 µl cells at a concentration of 2 x 10 6 cells / ml in complete medium. This gave a final concentration of 50 ng / ml PMA and 2 x 10 5 cells / well. The cells were allowed to spontaneously settle and the degree of aggregation was recorded at different times. Results ranged from 0-5 +, where 0 indicates that essentially no cells were found in clusters; 1+ indicated that not more than 10% of cells were in aggregates; 2+ indicated that no more than 50% of the cells were pooled; 3+ indicated that up to 100% of cells were in small, loose clusters; 4+ indicated that up to 100% of the cells were assembled in larger clusters; 5 and 5+ indicated that 100% of the cells were in large very compact aggregates. To obtain a more quantitative estimate of cellular adhesion, reagents and cells were added to 5 ml polystyrene tubes in the same manner as above. The tubes were placed in a rack on a rotary shaker at 37 ° C. After an hour at approx. 200 rpm 10 µΐ of the cell suspension was placed in a hemocytometer and the number of free cells determined. Percent aggregation was estimated by the following equation: χ15% aggregation = 100 x (1 - number of free cells \ number of cells added /

The number of cells added in the above formula is the number of cells per ml in a control tube containing only 20 cells and complete medium that has not been incubated. The number of free cells in the above equation corresponds to the number of non-aggregated cells per ml from experimental tubes. The above method is described by Rothlein, R., et al., J.Exper. With.

163: 1132-1149 (1986).

Example 3

LFA-1-dependent cellular aggregation.

The qualitative aggregation test described in Example 2 was performed using the Epstein-Barr transformed cell line JY. When adding PMA to the culture medium in the microtiter plates, aggregation of cells was observed. Time-shortened video recording showed that JY cells at the bottom of the microtiter wells were movable and exhibited active membrane ripple and pseudopodia movement. Contact between neighboring pseudopodia often resulted in cell-cell adhesion. If 5 adherence was maintained, the cell contact area moved to the uropod. Contact could be maintained despite vigorous cell movements and cell movement in different directions. The primary difference between PMA-treated and untreated cells was found to be the stability of these contacts when first formed. With PMA, clusters of cells grow in size as additional cells are adhered to their periphery.

As another means of measuring adhesion, the quantitative sample described in Example 2. was used. Cell suspensions were shaken at 200 rpm. for 2 hours, transferred to a hemocytometer and cells not in aggregates were counted. 1 absence of PMA was 42% (standard deviation = 20%, N = 6) of JY cells in 20 aggregates after 2 hours, while JY cells incubated under identical conditions with 50 ng / ml PMA had 87% (standard deviation = 8% , N = 6) of the cells in aggregates. Kinetic studies of aggregation showed that PMA increased the velocity and magnitude of all time points tested (Fig. 3).

Example 4

Inhibition of cell aggregation using anti-LFA-1 monoclonal antibodies.

To investigate the effect of anti-LFA-1 monoclonal antibodies on PMA-induced cellular aggregation, such antibodies were added to cells incubated in accordance with the qualitative aggregation sample 41 of Example 2. The monoclonal antibodies were found to inhibit the formation of cell aggregates either in the presence or absence of PMA. Both the F (ab *) 2 and Fab 'fragments of monoclonal antibodies were able to inhibit cellular aggregation against 5 LFA-1's α chain. Where essentially 100% cells formed aggregates in the absence of anti-LFA-1 antibody, it was found that no more than 20% of cells were in aggregates when antibody was added. The results of this experiment are described by Rothlein, R, et al. (J. Exper. Med. 163: 1132-1149 (19Θ6).

Example 5

Cellular aggregation needs the LFA-1 receptor.

15 EBV-transformed lymphoblastoid cells were prepared from patients analogous to the method described in Example 1. Such cells were screened for monoclonal antibodies capable of recognizing LFA-1 and the cells were found to be LFA-1 deficient. .

The qualitative aggregation test described in Example 2 was used, using the aforementioned LFA-1 deficient cells. Such cells were not able to spontaneously form aggregates, even in the near room of PMA.

Example 6

The recording of ICAM-1.

The LFA-1 deficient cells of Example 5 were labeled with carboxyfluorescein diacetate (Patarroyo, M. et al., Cell. Immunol. 63: 237-248 (1981)). The labeled cells were mixed at a ratio of 1:10 with autologous or JY cells, and the percentage of fluorescein labeled in cells in the aggregate is determined according to the method of Rothlein, R. et al., J ♦ Exper. With. 163: 1132-1149 (19Θ6). The LFA-1 deficient cells were found to be able to coaggregate with LFA-1 expressing cells (Fig. 4).

To determine if LFA-1 was important only in aggregate formation or in their preservation, antibodies capable of binding to LFA-1 were added to the aforementioned preformed aggregates. The addition of antibody was found to strongly disrupt the preformed aggregation. Time-shortened video recording confirmed that addition of the monoclonal antibodies to preformed aggregates started bursting within 2 hours (Table 1). After addition of monoclonal antibodies to LFA-1, pseudopodial movements and shape changes in individual cells within the aggregate continued unchanged. Individual cells gradually disassociated from the periphery of the aggregate; after 8 hours, most cells were dispersed. when reversed in time-shortened video recording, the bursting of preformed aggregates with LFA-1 monoclonal antibodies was found to be equivalent to the aggregation process in the absence of LFA-1 monoclonal antibody.

DK 176020 B1 43

Table 1

Ability of Antl-LFA-1 monoclonal antibodies to disrupt preformed PMA-induced JY cells aggregates.

5

Attempt Acquire Record Registration 18 hours _2 hours8_-mAb_ + mAb_ 10 14+ 4+ l + b 2 3+ 4+ 1 + c 3 _5 + _5 + 1 + ^ _

Aggregation in the qualitative microtiter plate sample was visually recorded. With anti-LFA-1 present throughout the trial, aggregation was at most 1+.

20 a The amount of aggregation just before the addition of monoclonal antibody after 2 hours.

hTSl / ie + TS1 / 22.

CTS1 / 18th

25 dTSl / 22.

DK 176020 B1 44

Example 7.

The need for divalent ions for LFA-1 dependent aggregation.

LFA-1 dependent adhesions between cytotoxic 5 τ cells and target cells require the presence of magnesium (Martz, E. J. Cell. Biol ♦ 84: 584-598 (1980)).

PMA-induced JY cell aggregation was tested for divalent cation dependence. JY cells did not form aggregates (using the sample of Example 2) in medium 10 without calcium or magnesium ions. The addition of divalent magnesium supported aggregation at concentrations as low as 0.3 mM. The addition of calcium ions alone had little effect. However, calcium ions were shown to increase the ability of magnesium ions to support PMA-induced aggregation. When 1.25 mM calcium ions were added to the medium, magnesium ion concentration ratios down to 0.02 mM were shown to support the aggregation. These data show that the LFA-1 dependent aggregation of cells requires magnesium ions, and that 20 calcium ions, although inherently insufficient, may act synergistically with magnesium ions to allow aggregation.

Example 8 The isolation of hybridoma cells capable of expressing anti-ICAM-1 monoclonal antibodies.

Monoclonal antibodies capable of binding to ICAM-1 were isolated according to the method of Rothlein, R. et al., J. Immunol.

137: 1270-1274 (1966). 3 BALC / C mice were immunized intraperitoneally with EBV-transformed peripheral blood mononuclear cells from an LFA-1 deficient individual (Springer, TA et al ♦, J. Exper. Med. 160: 1901 45 DK 176020 B1 (1984 )). Ca. 107 cells in ml of RPMI 1640 medium were used for each immunization. Immunizations were administered 45, 29 and 4 days before spleen cells were removed from the mice to produce the desired hybridoma cell lines. On day 3 before the removal of the spleen cells, the mice received an additional 107 cells in 0.15 ml of medium (intravenously).

Isolated spleen cells from the above animals were fused with P3X7 3Ag6,653 myeloma cells at a ratio of 10: 4: 1 according to Galfre, G. et al., Nature 266: 550 (1977). Aliquots of the obtained hybridoma cells were introduced into 96-well microtiter plates.

The hybridoma supernatants were screened for inhibition of aggregation and one inhibitory hybridoma 15 (of over 600 wells tested) was cloned and subcloned by border dilution. This subclone was designated RR1 / II (hereinafter referred to as "RR1 / 1").

Monoclonal antibody RR1 / 1 was found to substantially inhibit the LFA-1-expressed cell-linile. JY's PMA-stimulated aggregation. The RR1 / 1 monoclonal antibody similarly inhibited aggregation, or slightly less than certain monoclonal antibodies to LFA-1-α or -β subunits. In contrast, control monoclonal antibody to HLA, which is expressed on a regular basis on JY cells, did not inhibit aggregation. The antigen bound by monoclonal antibody RR1 / 1 is defined as the intercellular adhesion molecule-1 (ICAM-1).

DK 176020 B1 46

Example 9.

Use of anti-ICAM-1 monoclonal antibodies to characterize the ICAM-1 molecule.

To determine the nature of ICAM-1 and, in particular, whether 5 iCAM-i was different from LFA-1, immunoprecipitated cell proteins using monoclonal antibody rri / 1. Immunoprecipitation is performed according to the method of Rothlein, R. et al., J. Immunol. 137: 1270-1274 (1986)). JY cells were lysed at 5 x 10 7 cells / ml in 1% Triton X-100, 0.14 m NaCl,

10 mM Tris, pH 8.0, with freshly added 1 mM phenylmethylsulfonyl fluoride, 0.2 units per ml of trypsin inhibitor aprotinin (lysis buffer) for 20 minutes at 4 ° C. The lysate was centrifuged at 10,000 x g for 10 min. and pre-clarified 15 with 50 µl of a 50% suspension of CNBr-activated glycine-quenched Sephrose C1-4B for 1 hour at 4 ° C. 1 ml of lysate was immunoprecipitated with 20 µl of a 50% suspension of monoclonal antibody RR1 / i coupled to Sepharose C1-4B (1 mg / ml) overnight at 4 ° C

20 (Springer, T.A. et al ♦, J. Exper. Med. 160: 1901 (1984)). Sepharose-bound monoclonal antibody was prepared using CNBr activation of Sepharose CL-4B in carbonate buffer according to the method of March, S. et al. (Anal. Blochem. 60: 149 (1974)).

Twenty-five washed immunoprecipitates were subjected to SDS-PAGE and silver staining according to the method of Morrissey, J.H. Anal. Biochem. 117: 307 (1981).

After eluting proteins with SDS sample buffer (Ho, MK et al., J. Biol. Chem. 258-636 (1983)) at 30 ° C, the samples were halved and subjected to electrophoresis (SDS-8% PAGE). under reducing (Fig. 5A) or non-reducing conditions (Fig. 5B). Bands with molecular weights of 50 kd and 25 kd corresponded to the heavy and light chains of immunoglobulins from the monoclonal antibody Sepharose (Fig. 5A, lane 3). Also, varying amounts of other bands in the 25-50 kd weight range were observed, but not in precipitates from shallow leukemia cells, which yielded only a 90 kd molecular weight band. LFA-1's I77kd α subunit and 95 kg β subunit were found to migrate differently from ICAM-1 under both reducing (Fig. 5A, lane 2) and non-reducing (Fig. 5B, lane 2) conditions.

In order to determine the effect of monoclonal antibody RR1 / l on PHA lymphoblast aggregation, the quantitative aggregation assay described in Example 2. T-cell blast cells were thus stimulated for 4 days with PHA, washed thoroughly and then cultured for 6 days 15 in the presence of il-2 conditioned medium. The PHA was found to be internalized during this 6 day cultivation and did not contribute to the aggregation test. In three different samples with different T cell blast preparation, ICAM-1 monoclonal antibody aggregation 20 was uniformly inhibited (Table 2).

DK 176020 B1 48

Table 2

Inhibition of PMA-stimulated PHA lymphoblast aggregation of RR1 / 1 monoclonal antibody8 5%%

Attempt PMA Mab_Aggregation Inhibition - Control 9 --- + Control 51 0 10 + HLA-A, B 58 -14d + LFA-1-α 31 39 + ICAM-1 31 39 2e - Control 10 --- + Control 78 0 15 + LFA-lp 17 78 + ICAM-1 50 36 3f - ----- 7 + Control 70 + HLA-Α, Β 80 -14 20 + LFA-3 83 -19 + LFA-1-α 2 97 + LFA -1-β 3 96 + ICAM-1 34 51 25

Aggregation of PHA-induced lymphoblasts stimulated with 50 ng / ml PMA was quantitated indirectly by microscopic counting of the number of non-aggregated cells as described in Example 2.

30 b Percent inhibition relative to cells treated with PMA and X63 monoclonal antibody.

Aggregation was measured 1 hour after the simultaneous addition of monoclonal antibody and PMA to the 49 'DK 176020 B1. Cells were shaken at 175 rpm.

^ A negative number indicates percent aggregation increase.

Aggregation was measured 1 hour after the simultaneous addition of a monoclonal antibody and PMA. Cells were pelleted at 200 x G for 1 min, incubated at 10 37 ° C for 15 min, gently resuspended and shaken for 45 min. at 100 rpm

Cells were pretreated with PMA for 4 hours at 37 ° C. After monoclonal antibody was added, the tubes were incubated at 37 ° C stationary for 20 min. and shaken at 75 rpm. for 100 min.

B1 LFA-1 monoclonal antibodies were consistently more inhibitory than iCAM-1 monoclonal antibodies, whereas HLA-A, B and LFA-3 monoclonal antibodies were ineffective. These results indicate that of the monoclonal antibodies present, only those capable of binding to LFA-1 or ICAM-1 were able to inhibit cellular adhesion.

Example 10 10 Preparation of monoclonal antibody to ICAM-1.

Immunization.

A Balb / C mouse was immunosuppressed intraperitoneally (i.p) with 0.5 ml of 2 x 10 7 JY cells in RPMI medium 103 15 days and 24 days before fusion. On the 4th and 3rd day before fusion, mice were immunized i.p. with 107 cells of PMA-differentiated U937 cells in 0.5 ml of RPMI medium.

Differentiation of U937 cells.

20 U937 cells (ATCC CRL-1593) differentiated by incubating these at 5 x 10 5 / ml in RPMI with 10% fetal bovine serum, 1% glutamine and 50 µg / ml gent amyin (complete medium) containing 2 ng / ml phorbol -12-myristate acetate (PMA) in a sterile polypropylene container.

On the third day of this incubation, half of the volume of the medium was removed and replaced with fresh, complete medium containing PMA. On day 4, cells were removed, washed and prepared for immunization.

30 Fusion.

Spleen cells from the immunized mice were fused with P3x63 Ag8,653 myeloma cells at a 4: 1 ratio according to Galfre et al. > (Nature 266: 550 51 DK 176020 B1 (1977)). Following the fusion, the cells were plated in a {96 well flat bottom microtiter plate at 510 spleen cells / well.

5 Selection of anti-ICAM-1 positive cells.

After one week, 50 µl of supernatant screening was subjected to the qualitative aggregation test of Example 2, using both JY and SKW-3 as aggregating cell lines. Cells from supernatants that inhibit JY cell aggregation but not SKW-3 were selected and cloned twice relieving limiting dilution.

This experiment resulted in the identification and cloning of three separate hybridoma alleles producing 15 anti-ICAM-1 monoclonal antibodies. The antibodies produced by these hybridoma alleles were IgG2a, IgG2b and IgM, respectively. The hybridoma cell line that produced the IgG2a anti-ICAM-1 antibody is assigned the designation R6 D6 E9 B2. The antibody produced by the preferred hybridoma cell line was designated R6 D6 E9 B2 (hereinafter referred to as "Re-S-De"). x Hybridoma cell line R6 5 D6 E9 B2 was deposited with the American Type Culture Collection on October 30, 1987 and awarded the designation ATCC HB 9580.

25

Example 11! The expression and regulation of ICAM-1.

To measure ICAM-1 expression, a radioimmunoassay was developed. In this sample, purified -30 RRl / 1 was iodinated using lodogen for a specific activity of 10 µCi / µg. Endothelial cells were cultured in 96 well plates and treated as described for each experiment. The plates were cooled to 4 ° C by application in a cold room for 0.5-1 hour, Not directly on ls. The monolayers were washed 3 times with cold, complete media and then incubated for 30 min. at 4 ° C with 125 I RR1 / 1. The monolayers were then washed 3 times 5 with complete media. The bound was released using 0.1 N NaOH and counted. The specific activity of 125 I RR1 / 1 was adjusted, using unlabeled RH1 / 1, to obtain a linear signal within the antigenic densities encountered during this study. Non-specific binding was determined in the presence of a thousand-fold excess of unlabeled RR1 / 1 and subtracted from the total binding to obtain the specific binding.

XCAM-1 expression, measured using the above-mentioned radloimmune sample, is increased on human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) of IL-1, TNF, LPS and IPN-γ (Table 3). Saphenous vein endothelial cells were used in this study to confirm the results of umbilical vein endothelial cells in cultured large venous endothelial cells from fully developed tissue. The basal expression of ICAM-1 is 2-fold higher on saphenous vein endothelial cells than on umbilical vein endothelial cells. Exposure of the umbilical vein endothelial cell. for recombinant IL-1-25 α, ιι, -1-β and TNF-γ increases ICAM-1 expression 10-20 fold.

IL-1-α, TNF, and LPS were among the most potent inducers and IL-1 was less potent on a weight basis and additionally at saturation concentrations for response (Table 3).

IL-1-β at 100 ng / ml increased ICAM-1 expression 9-fold on HUVEC and 7.3-fold on HSVEC with half-maximal increase occurring at 15 ng / ml. rTNF at 50 ng / ml increased lCAM-1 expression 16-fold on HUVEC and 11-fold on HSVEC with half-maximal effects at 0.5 ng / ml.

53 DK 176020 B1

Interferon-γ produced a significant increase in ICAM-1 expression of 5.2-fold on HUVEC or 3.5-fold on HSVEC at 10,000 U / ml. The effect of LPS at 10 µg / ml was of the same order of magnitude as rTNF.

Pairwise combinations of these mediators resulted in additional or slightly less than additional effects on ICAM-1 expression (Table 3). Cross-titration of rTNF with rIL-1-β or rIFN-γ showed no synergism between these at suboptimal or optimal concentrations.

Since LPS increased ICAM-1 expression on endothelial cells at concentrations sometimes found in culture media, it was investigated the possibility that basal ICAM-1 expression may be due to LPS. When several 15 serum batches were tested, low endotoxin serum was found to give lower ICAM-1 basal expression at 25%. All of the results set forth herein were for low endotoxin serum culture. However, the inclusion of the LPS-neutralizing antibiotic polymyxin B at 10 µg / ml 20 only reduced the ICAM-1 expression by an additional 25% (Table 3). The increase in ICAM-1 expression by treatment with IL-1 or TNF was not affected by the presence of 10 µg / ml polymyxin B, which is consistent with the low endotoxin concentrations in these 22f preparations (Table 3).

DK 176020 B1 54 '

Table 3

Anti-iCAM-1 monoclonal antibodies.

5 'Condition (16 hours) + 1 Specific Bound (CPM)

HSVEC HSVEC

l-Control 603 + 11 - 1132 + 31 - 100 ng / ml rIL-1 beta ·. 5680 ~ ± 633 9x 8320 + 766 7.3x _ 10 50 ng / ml rIL-1 alpha 9910 + 538 16x 50 ng / ml rTNF alpha 1 9650 + 1500 16x 12690 + 657 11.2x.

10 µg / ml LPS 9530 ± 512 16x 10459 ± 388 9.2x 10 ng / ml rlFN gamma 3120 + 308 5.2x 4002 + 664 3.5x 'rIL-1 beta + rTNF 1469 + 1410 24x 16269 + 660 14x · rIL-1 beta + LPS 13986 "+ 761 23x 10870 + 805 10x 15 rIL-1 beta + rlFN gamma 7849 + 601 13x 8401 + 390 7.4x rTNF + LPS 15364 '+ 1241 24x 16141 + 1272 14x rTNF + rlFN gamma 13480 + 1189 22x 13238 + 761 12x, LPS + IFN gamma 10206 + 320 17x 10987 + 668 10x polymyxin B (10 pg / ml) 480 + 23 polymyxin B + rIL-1 15390 + 97 lix -, λ polymyxin B + rTNF .9785 + 389 20x 1 ^ g / ml LPS 17598 + 432 13x polymyxin B + LPS 10510 + 44 l.lx

I M

in

Up-regulation of ICAM-1 expression on HVEC and HSVEC-25 HUVEC or HSVEC was seeded in 96-well plates at a 1: 3 from a liquid monolayer and allowed to grow to confluence. The cells were then treated with the indicated materials or media for 16 hours and RIA as per methods. All points were completed 4 30 times.

in DK 176020 B1 55

Example 12

Kinetics of Interleukin 1 and γ-interferon induction of ICAM-1.

The kinetics of interleukin 1 and γ interferon-5 effects on ICAM-1 expression on dermal fibroblasts was determined using the 125 I goat anti-mouse IgG binding assay by Dustin, M.L. et al.

(J. Immunol. 137: 245-254 (1986). To carry out this binding assay, human dermal fibroblasts were grown in a 96-well ml crotiter plate to a density of 2-8 x 10 4 cells / well (0.32 The cells were washed twice with RPMI 1640 medium supplemented as described in Example 1. The cells were washed once more with Hank's balanced saline (HBSS), 10 mM HEPES, 0.05% NaN3, and 10% heat-inactivated fetal bovine serum, washing. with this binding buffer was carried out at 4 ° C.

To each well, 50 μΐ of the above binding buffer and 50 μΐ of the appropriate hybridoma supernatant with X63 and W6 / 32 were added as the negative and positive 20 controls, respectively. After incubation for 30 minutes at 4 ° C with gentle stirring, the wells were washed twice with binding buffer, and the second antibody 125 I goat anti-mouse IgG was added at 50 nCi for 100 μΐ. The 125 I goat anti-mouse antibody was prepared using iodogen (Pierce) according to the method of Fraker, P.J. et al. (Biochem. Blophys. Res. Commun.

80: 849 (1978)). After 30 min. at 4 ° C, the wells were washed twice with 200 μΐ binding buffer and the cell layer was dissolved by adding 100 μΐ 0.1 N NaOH. This and a 30 loo μΐ wash were counted in a Beckman 5500 γ counter. The specific number of counts per min. was calculated as [cpm with monoclonal antibody] - [cpm with X63]. All steps, including specific reagent induction, were performed 4 times.

56 DK 176020 B1

The effect of interleukin 1 with a half-life of 1 CAM-1 induction of 2 hours was faster than the effect of γ-interferon with a half-life of 3.75 hours (Fig. 6). The time course for returning to residual ICAM-1 concentrations was found to be dependent on the cell cycle or rate of cell growth. In quiescent cells, interleukin 1 and γ interferon effects are stable for 2-3 days, whereas ICAM-1 expression depicted as log phase cultures is 10 near baseline 2 days after removal of these inducers.

Dose-response curves for the induction of ICAM-1 by recombinant mouse and human interleukin 1 and for recombinant human γ-interferon are shown in FIG. 7. γ-15 interferon and interleukin 1 were found to have similar concentration dependencies with nearly identical effects at 1 ng / ml. In addition, human and recombinant mouse interleukin 1 had similar curves but are less effective than human interleukin 1 preparations in inducing ICAM-1 expression.

Cyclohexamide, an inhibitor of protein synthesis, and actinomycin-D, an inhibitor of mRNA synthesis, abolish the effects of both interleukin 1 and γ-interferon on ICAM-1 expression on fibroblasts (Table 4).

Tunicamycin, an inhibitor of N-linked glycosylation, further inhibited only interleukin 1 activity by 43%. These results indicate that protein and mRNA synthesis but not N-linked glycosylation is required for interleukin 1- and γ-interferon-stimulated increases in ICAM-1 expression.

DK 176020 B1 57 TABLE 4

Effects of cycloheximide, actinomycin D and tunica-mycin on ICAM-1 induction with IL-1 and γ-IFN on human dermal fibroblasts®.

5 125, Goat Anti-Mouse IgG (Specifically Bound icpm)

Behandling_anti-Icah-1_antl-HLA-A.B.C

Control (4 hours) 1524 4 140 11928 ♦ 600 hours cycloheximide 1513 ± 210 10678 ± 471 4 actinomycin 0 1590 ± 46 12276 4 608 4 tunlcamycline 1461 ± 176 12340 4 940 IL 1 (10 0 / ml) (4 hours) 4264 4 249 12155 4 510 4 cycloheximide: 1619 4 381 12676 4 446 15 4 actinomycin D 1613 4 88 12294 ± 123 4 tunicamycin 3084 4 113 13434 4 661 I FN-γ (10 U / ml) (18 hours) 4659 4 109 23675 ± 500 4 cycloheximide 1461 4 59 10675 4 800 4 actinomycin D 1326 ± 186 12089 4 550 20 AHuman fibroblasts were grown to a density of 8 x 10 4 cells / 0.32 cm 2 well. Treatments were carried out in a final volume of 50 μΐ containing the indicated reagents.

25 cycloheximide, actinomycin D and tunicamycin were added in 20 µg / ml, 10 µM and 2 µg / ml, respectively, simultaneously with the cytokines. All points are mean values from four farmers + standard deviation.

·.

DK 176020 B1 58

Example 13 ICAM-11's tissue distribution.

Histochemical studies were performed on frozen tissues from human organs to determine the distribution of 5 ICAM-1 in the thymus, lymph nodes, intestine, skin, kidneys and liver. To perform such an analysis, frozen tissue pieces (4 µm thick) of normal human tissue were fixed in acetone for 10 min. and stained with the monoclonal antibody RR1 / 1 by an immunoperoxidase technique, using the avidin-biotin complex method (Vector Laboratories, Burlingame, CA) described by Cerf-Bensussan, N. et al. (J. Immunol. 130: 2615 (1983)). After incubation with the antibody, the pieces were incubated continuously with biotinylated equine anti-mouse IgG and avidin-biotinylated peroxidase complexes. The pieces were finally dipped in a solution containing 3-amino-9-ethyl-carbazole (Aldrich Chemical Co., Inc., Milwaukee, Wl) to develop a color reaction. The pieces were then fixed in 4% formaldehyde for 5 minutes. and counterstained with 20 hematoxylin. Check included pieces Incubated with unrelated monoclonal antibodies instead of the RR1 / 1 antibody.

ICAM-1 was found to have a distribution substantially equal to that of the majority of the 25 histocompatibility complex (MHC) class II antigens. Most blood vessels (both small and large) in all tissues showed staining of endothelial cells with ICAM-1 antibody.

The vascular endothelial staining was more intense in the interfollicular (paracortical) areas of lymph nodes, 30 tonsils, and Peyer's "patches" compared to vessels in the kidney, liver and normal skin. In the liver, staining was essentially confined to sinusoidal garment cells; the hepatocytes and endothelial cells, inside most of the portal veins, and the arteries were not stained.

In the thymus marrow, diffuse staining of large cells and a dentritic staining pattern were observed. In cort-5 ex, the staining pattern was focal and essentially dentritic. Thymocytes were not stained. In the peripheral lymphoid tissue, the germinal center cells of the secondary lymphoid follicles were intensely stained. In some lymphoid follicles, the staining pattern was essentially dentritic without recognizable staining of lymphocytes.

In addition, weak staining of cells in the sheath zone was observed. Dendritic cells with cytoplasmic extensions (interdigitating reticulum cells) and a small number of lymphocytes in the interfollicular or.

Fifteen paracortical regions were further stained with ICAM-1 binding antibody.

Cells similar to macrophages were stained in the lymph nodes and small intestinal lamina propria. Fibroblast-like cells (spindle-shaped cells) and detritic cells scattered in the stroma and most of the organs examined are stained with the iCAM-1 binding antibody. No staining was observed in the Langerhaan / indeterminate cells of the epidermis. No staining in smooth muscle tissue was found.

25 The staining of epithelial cells could similarly be seen in the mucosa of the tonsils. Although in most cases hepatocytes, bile duct epithelium, intestinal epithelial cells, and tubular epithelial cells are not stained, pieces of normal kidney tissue obtained from a nephrectomy with renal cell carcinoma showed staining of many proximal tubular. cells for ICAM-1. These tubular epithelial cells are additionally stained with an anti-HLA-DR binding antibody.

60 DK 176020 B1

In summary, ICAM-1 is expressed on non-hematopoietic cells such as vascular endothelial cells and on hematopoietic cells such as tissue macrophages and mitogen-stimulated T lymphocyte hybrids. ICAM-1 was found to be expressed in small amounts on peripheral blood lymphocytes.

Example 14

The purification of ICAM-1 by monoclonal antibody affinity chromatography 10

General cleaning schedule.

ICAM-1 was purified from human cells or tissues using monoclonal antibody affinity chromatography. Monoclonal antibody, RRI / 1, reactive to ICAM-1 was first purified and coupled to an inert column matrix. This antibody is described by Rothlein, R Si · J · Immunol. 137: 1270-1274 (1986), and Dustin, M.L. et al. (J. Immunol. 137: 245 (1986). ICAM-1 was solubilized from cell membranes by lysing cells 20 in a non-ionic detergent, Triton X-100, near a neutral pH. The cell lysate containing dissolved ICAM-1 then through pre-columns destined for removal of non-specific binding material to the column matrix material, and then through the monoclonal antibody column matrix enabling ICAM-1 to bind to the antibody. The antibody column was then washed with a series of detergent washing buffers. with increasing pH up to pH 11.0 During these washes, ICAM-1 remained bound to the antibody matrix while removing 50 non-binding and weakly binding contaminants. The bound ICAM-1 was then specifically eluted from the column by application of a detergent buffer of pH 12.5.

61 DK 176020 B1

Purification of monoclonal antibody RR1 / 1 and covalent coupling to Sepharose CL-4B.

The anti-ICAM-1 monoclonal antibody RRi / l was purified from asymmetric fluid from hybridoma-bearing mice, or from hybridoma culture supernatants by standard ammonium sulfate precipitation and protein A affinity chromatography techniques (Ey et al., Immunochem. 15: 429 1978)). The purified IgG, or rat IgG (Sigma Chemical Co., St. Louis, MO) was covalently coupled to I Sepharose CL-4B (Pharmacia, Upsala, Sweden) using a modification of the method of March et al. (Anal. Blochem. 60: 149 (1974)). Briefly, Sepharose CL-4B was washed in distilled water, activated with 40 mg / ml CNBr in 5 M K 2 HPO 4 (pH approx. 12) for 5 min. and then washed extensively with 0.1 mM HCl at 4 ° C. The filtered activated Sepharose was resuspended with a corresponding amount of purified antibody (2-10 mg / ml in 0.1 M NaHCO 3, 0.1 M NaCl). The suspension was incubated for 18 hours at 4 ° C with gentle "end-over-20" rotation. The supernatant was then checked for unbound antibody by absorbance at 280 nm, and residual reactive sites on activated Sepharose were saturated by adding glycine to 0.05 M. The coupling efficiency was usually above 90%.

25

Detergent solubilization of membranes prepared from human spleen.

All procedures were carried out at 4 ° C. Frozen human spleen (200 g fragments) from patients with 30 hair cell leukemia were thawed on ice in 200 ml Tris saline solution (50 mM Tris, 0.14 M NaCl, pH 7.4 at 4 ° C) containing 1 mM phenylmethylsulfonyl fluoride ( PMSF), 0.2 U / ml aprotinin and 5 mM iodoacetamide. The tissue was cut 62 · DK 176020 B1 into small pieces and homogenized at 4 ° C with a Tekmar power homogenizer. The volume was then increased to 300 ml with Tris saline solution and 100 ml of 10% Tween 40 (polyoxyethylene sorbitan monopalmitate) in Tris saline solution was added to give a final concentration of 2.5% Tween 40.

To make membranes, homogenate one was extracted with three strokes from a Dounce or, more. Preferably, a Teflon Potter Elvejhem homogenizer 10 µg was then centrifuged at 1000 x g for 15 minutes. The supernatant was retained and the pellet was re-extracted with 200 ml of 2.5% Tween 40 in Tr ice-sal top solution. After centrifugation at 1000 x g for 15 minutes, the supernatants were collected from both extractions and centrifuged at 150,000 x g for 1 hour to pellet the membranes.

The membranes were washed by redissolving in 200 ml Trissalt solution, centrifuged at 150,000 x g for 1 hour. The membrane pellet was resuspended in 200 ml of Tris salt solution and homogenised with a motorized homogenizer and a Teflon pestle until our suspension in evenly cloudy, the volume was then increased to 900 ml with

Tris saline solution and N-lauroylsarcosine were added to a final concentration of 1%. After stirring at 4 ° C for 30 min. insoluble material in detergent lysate 25 was removed by centrifugation at 150,000 x g for 1 hour. Triton X-100 was then added to the supernatant to a final concentration of 2% and the lysate was stirred at 4 ° C for 1 hour.

Detergent solubilization of JY B lymphoblast cells.

The EBV-transformed B lymphoblastoid cell line JY was grown in RPMI-1640 containing 10% fetal calf serum (FCS) and 10 mM HEPES to a density of 63.

DK 176020 B1 approx. 0.8-1.0 x 10 6 cells / ml. To increase cell surface expression of ICAM-1, phorbol-12-myristate-13-acetate (PMA) was added at 25 ng / ml 8-12 hours before harvesting of the cells. In addition, sodium vanadate (50 μΜ) was added to the cultures at this time. The cells were pelleted by centrifugation at 500 x g for 10 min, and washed twice in Hank's Balanced Saline (HBSS) by resuspending and centrifugation. The cells (approximately 5 g per 5 l of culture) were lysed in 50 ml of lysis buffer (0.14 M NaCl, 1Θ50 mM Tris pH 8.0, 1% Triton X-100, 0.2 U / ml aprotinin, 1 mM PMSF (50 μΜ sodium water) with stirring at 4 ° C for 30 min. Non-lysed nuclei and insoluble material were removed by centrifugation at 10,000 x g for 15 min. followed by centrifuging the supernatant 15 at 150,000 x g for 1 hour and filtering the supernatant through Whatman 3 mm filter paper.

Affinity chromatography of ICAM-1 for structural studies.

For large-scale purification of ICAM-1 to be used in structural studies, a column of 10 ml of RR1 / 1-Sepharose CL-4B (coupled with 2.5 mg of antibody / ml of gel) was used and two 10 ml of Columns with CNBr-activated, glycine-quenched Sepharose CL-4B and rat-2β IgG coupled to Sepharose CL-4B (2 mg / ml) were used. The columns were serially connected and pre-washed with 10 column volumes of lysis buffer, 10 column volumes buffer of pH 12.5 (50 mM triethylamine, 0.1% Triton X-100, pH 12.5 at 4 ° C, followed by equilibration with ^ 10 column volumes One liter of human spleen detergent lysate was supplied at a flow rate of 0.5-1.0 ml per minute. The two pre-columns were used to remove non-specific binding material from the lysate prior to passage through RR1 / l. -Sepharose column.

64 DK 176020 B1

After the addition, the RR1 / 1-Sepharose column was washed and bound ICAH-1 continuously at a flow rate of 1 ml / min. with at least 5 column volumes of each of the following: 1) lysis buffer, 2) 20 mM 5 Tris pH 8.0 / 0.14 M NaCl / 0.1% Triton X-100, 3) 20 mM glycine pH 10.0 / And 4) 50 mM triethylamine pH 11.0 / 0.1% Triton X-100. All wash buffers p contained 1 mM PMSF and 0.2 U / ml aprotinin. After washing, the remaining bound icam-i was eluted with 10 5 column volumes of elution buffer (50 mM triethylamine / 0.1% Triton X-100 / pH 12.5 at 4 ° C) at a flow rate of 1 ml / 3 mine. The eluted ICAM-1 was collected in 1 ml fractions and immediately neutralized by the addition of 0.1 ml of 1 M Tris, pH 6.7. Fractions 15 containing ICAM-1 were identified by SDS-polyacrylic amide electrophoresis of 10 μΐ aliquots (Springer et al., J. Exp. Med. 160: 1901 (1984)), followed by silver staining (Morrissey, JH, Anal. Blochem. 117 : 307 (1981)).

Under these conditions, the amount of ICAM-1 was eluted 20 for approx. 1 column volume more than 90% purely judged from silvery electropherograms (a small amount of IgG, leached from the affinity matrix, was the major polluter). The fractions containing ICAM-1 were pooled and concentrated to ca. 20 times using 25 Centricon-30 microconcentrators (Amicon, Danvers, MA). The purified ICAM-1 was quantified by Lowry protein sample of an ethanol-precipitated aliquot from the pool: Approx. 500 µg pure ICAM-1 was produced from 200 g human spleen.

30 Approx. 200 µg of purified ICAM-1 was exposed to one

second-stage purification by preparative SDS-polyacrylamide gel electrophoresis. The band representing ICAM-1 was made visible by soaking the gel in 1 M

DK 176020 B1 | 65 · KCl. The gel region containing ICAM-1 was then excised and electroeluted in accordance with the method of Hunkapiller et al., Meth. Enzymol. 91: 227-236 (1983). The purified protein was more than 98% 5 purely assessed by SDS-PAGE and silver staining.

Affinity purification of ICAM-1 for functional studies.

ICAM-1 for use in functional studies was purified from detergent lysates of JY cells as described above, but on a smaller scale (a 1 ml column of RR1 / i-Sepharose) and with the following modifications.

All solutions contained 50 μΜ sodium vanadate. After washing the column with pH I, O buffer containing 0.1% Triton X-100, the column was washed again with five 15 column volumes of the same buffer containing 1% n-actyl-pD-glucopyranoside (octylglucoside) instead of 1. 0% Triton X-100. The octylglucoside detergent displaces Triton X-100 bound to ICAM-1 and, unlike Triton X-100, it can later be removed by dialysis.

20 ICAM-1 was then eluted with pH 12.5 buffer containing 1% octyl glucoside instead of 0.1% Triton X-100 and analyzed and concentrated as described above.

Example 15 Characteristics of Purified ICAM-1.

Human spleen ICAM-1 purified in SDS-polyacrylamide gels migrates as a broad band with Mr of 72,000 to 91,000. ICAM-1 purified from JY cells also migrates as a broad band with Mr of 76,500 to 97,000. These Mr 30 are within the indicated range of ICAM-1 immunoprecipitated from various cell sources: Mr = 90,000 for JY cells, 114,000 on the myelomonocytic cell line U937 and 97,000 on fibroblasts (Dustin et al., J.

DK 176020 B1 I 66! Immunol. 137: 245 (1986)). This broad area in Mr to is written an extensive but variable degree of glyco-cycling. The non-glycosylated precursor has a Mr of 55,000 (Dustin et al.). The protein purified from either 5 JY cells or human spleen retains its antigenic effect, evidenced by its ability to bind to the original affinity column and by immunoprecipitation with RR1 / 1-Sepharose and SDS-polyacrylamide electrophoresis.

For the preparation of peptide fragments of ICAM-1, ca. 200 µg with 2 mM dithiothreitol / 2% SDS, followed by alkylation with 5 mM iodoacetic acid. The protein was precipitated with ethanol and redissolved in 0.1 M NH 4 CO 3 / 0.1 Mm CaCl 2 / 0.1% zwittergen 3-14 (Calbio-13 chem) and subjected to digestion with 1 wt% trypsin at 37 ° C for 4 hours. followed by further digestion with 1% trypsin for 12 hours at 37 ° C. The tryptic peptides were purified by reverse-phase HPLC using a 0.4 x 15 cm C4 column (Vydac). The peptides were eluted in a linear gradient of 0% to 60% acetonitrile in 0.1% trifluoroacetic acid. Selected peptides were subjected to sequence analysis on an Applied Biosystems gas phase micro-equator. The sequence information obtained from this study is shown in Table 5.

DK 176020 B1 67

Table 5

Amino acid sequences of ICAM-1 tryptic peptides.

Amtno- Peptide 5 Acid -____

Sfi8 * -...... 50a SOb 46a 46b X 4S K AA J U 0 Ml

1 (T / V) A (V / A) E VS LE A L V L

2F SQ P-EFNLSITl / E

3 1 IT ALPPDS6L P / (6)

4 7 $ F AATLV1 P

10 SVIP P P V R l E G / Y

6Y GL LNTP VT N / L

7 P W P P V Y Q T P (N).

8 T P 1 I T / l G G C P / V (Q) 8 S F G (G) I - I S K (E)! E i (Q) - DE T (0)

11 A S D / P K $ L S

15 12 G / S V V P F F C

I 13 A T O Q S E D

14 G V V V / L A - Q

15 I K T P

16 S K

17 A

18 p

19 X

20 20 O.

21 L

() s Low Confidence Sequence.

[] - Very low conflicts sequence.

25 / s indicates ambiguity in the sequence; the most likely amino acid is listed first, a s Larger peptide, b = Less peptide.

DK 176020 B1; 68

Example 16

Cloning of the ICAM-1 gene.

The gene for ICAM-1 can be cloned peaks using a variety of methods. Eg. For example, the amino acid sequence information obtained through the sequencing of the tryptic fragments of ICAM-1 (Table 5) can be used to identify an oligonucelotide sequence that will correspond to the ICAM-1 gene. Alternatively, the ICAM-1 gene can be cloned using anti-ICAM-1 antibody 10 to detect clones producing ICAM-1.

Cloning of the ICAM-1 gene through the use of oligonucleotide probes ♦

Using the genetic code (Watson, 15 JD, In: Molecular Biology of the Gene, 3rd ed., WA Benjamin, Inc., Menlo Park, CA (1977)), one or more different oligonucleotides can be identified, each of which will be able to encode the ICAM-1 tryptic peptides. The likelihood that a particular 20 oligonucleotide actually constitutes the current ICAM-1 coding sequence can be estimated by considering abnormal base-pair relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon 25 codes of conduct" are set forth by Lathe, R., et al., J.

Molec. Biol. 183: 1-12 (1985). Using the "codon cutaneous rules" of Lathe, a single oligonucleotide, or a set of oligonucleotides, containing a theoretical "most likely" nucleotide sequence (i.e., the least excess nudeotide sequence) is identified which is capable of to encode the ICAM-1 tryptic peptide sequences.

The oligonucleotide or set of oligonucleotides containing the theoretical "most likely" sequence capable of encoding the ICAM-1 fragments is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing the "most likely 5" sequence, or set of sequences. An oligonucleotide containing such a complementary sequence can be used as a probe to identify and isolate the ICAM-1 gene (Maniatis, T., et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, 10 Cold Spring Harbor, NY ( 1982).

As described in Section C above, it is possible to clone the ICAM-1 gene from eukaryotic DNA preparations which are believed to contain this gene. For identification and cloning of the gene encoding the ICAM-1 protein, a DNA library is subjected to its ability to hybridize with the aforementioned oligonucleotide probes.

Since it is likely that there will only be two copies of the gene for ICAM-1 in a normal diploid cell and because it is possible that this ICAM-1 gene may contain large 20 non-transcribed intermediate sequences (introns) if cloned not desired, it is desirable to isolate ICAM-1 coding sequences from a cDNA library prepared from mRNA from an ICAM-1 producing cell rather than from genomic DNA. Suitable DNA or cDNA preparations are enzymatically cleaved or clipped and ligated into recombinant vectors.

The ability of these recombinant vectors to hybridize to the above oligonucleotide probes is then measured. Methods of hybridization are indicated e.g. in 30 Maniatis, T., Molecular Cloning A Laboratory Manual,

Cold Spring Harbor Press, Cold Spring Harbor, NY (1982) or in Haymes, B. T., et al., Nucleic Acid Hybridization and Practiclal Approach, IRL Press, Oxford, England in 70 DK 176020 B1 (1985). Vectors which were found to be capable of such hybridization are then analyzed to determine the extent and nature of the ICAM-1 sequences which they contain. Based on purely statistical considerations, a gene such as that encoding the ICAM-1 molecule could be uniquely identified (via hybridization screening) using an oligonucleotide probe of only 18 nucleotides.

Thus, the current identification of iCAM-1 peptide 10 sequences allows, in short, the identification of a theoretically "most likely" DNA sequence or set of such sequences capable of encoding such a peptide. oligonucleotide complementary to this theoretical sequence 15 (or by constructing a set of oligonucleotides complementary to the set of the "most probable" oligonucleotides) yields a DNA molecule (or set of DNA molecules) capable of act as a probe to identify and isolate the ICAM-1 gene.

Using the ICAM-1 peptide sequences listed in Table 5, the sequence of the "most likely" sequence was determined by an oligonucleotide capable of encoding AA and J peptides (Tables 6 and 7, respectively). Oligonucleotides complementary to these sequences were synthesized and purified for use as probes to isolate ICAM-1 gene sequences. Suitable size-selected cDNA libraries were produced from poly (A) + RNA from both PMA-induced HL-60 cells and from PS-stimulated umbilical vein endothelial cells. A 30 size-selected cDNA library was prepared using poly (A) + RNA from PMA-induced HL-60 cells according to the method of Gubier, U., et al. ((Gene 25: 263-269 (1983)) and Corbi, A-, et al (EMBO J. 6: 4023-4028 (1987)).

in DK 176020 B1 i 71 ·

A site-selecting cDNA library was prepared using poly (A) + RNA from umbilical vein endothelial cells that had been stimulated for 4 hours with PS 5 µg / ml. RNA was extracted by homogenizing the 5 cells in 4 M guanidinium isothiocyanate and the supernatant was subjected to ultracentrifugation through a CsCl gradient (Chirgwin, J. M., et al., Blochem. 18: 5294-5299 (1979)). Poly (A) + RNA was isolated from the mixture of total RNA pieces via the use of oligo-10 (dT) cellulose chromatography (Type 3, Collaborative Research) Aviv, H., et al., Proc. Natl. Acad. Sci. (USA) 69: 1408-1412 (1972).

i i! 72 DK 176020 B1

Table 6

Oligonucleotide complementary to the most likely nucleotide sequence capable of encoding the ICAM-1-AA peptide.

5 _

Amino ICAM-1 Most Likely Complementary Acid Residue AA Peptide Sequence Encoding Sequence of ICAM-1 for AA Peptide T '5 »y

in 162 GI u G C

υ A T

G C

163 Leu C G

T A

G C

164 Asp G C

15 A T

15 in C G

165 Leu C G

T A

G C

, 166 Arg C G

G C

in G C

20 167 Pro C G

C G

C G

168 Gin C G

A T

G C

in 169 Gly G C

25 I j j

I c G

: 170 Leu C G

T A

G C

: 171 Glu G C

AT G C

30: 172 Leu C G

! T A

G C

173 Phe T A

T A

T A

174 Glu G C

A T

G C

I ~ ....... 3 '5' 73 DK 176020 B1

Table 6 (continued)

Oligonucleotide complementary to the most likely nucleotide sequence capable of encoding the ICAM-1-AA peptide.

5 · _ in 1 ♦ - Amino ICAM-1 Most Likely Complementary Acid Residue AA Peptide Sequence Encoding Sequence of ICAM-1 for AA Peptide 10

175 Asn A T

A T

C 6

176 Thr A T

C 6 C 6

15 177 See U A

C G

A

3 '5' in DK 176020 B1 74

Table 7

Oligonucleotide complementary to the most likely nucleotide sequence capable of encoding the ICAM-1-J peptide.

5______

Amino ICAM-1 Most Likely Complementary Acid Residue AA Peptide Sequence Encoding Sequence of ICAM-1 for AA Peptide 5 '3'

10 19 Val 6 C

T A

G C

Thr A T

C G

C G

21 Cys T A

G C

15 CG

22 Ser T A

C G

C G

23 Thr AT

C G

C G

20 24 Ser T A

C G

C G

I 25 Cys T A

G C

T A

26 Asp G C

25 AT

C G

27 Gin C G

A T

G C

28 Pro C G

C G

C G

30 29 List A T

A T

3 '5' 75 · DK 176020 B1 First cDNA strand was synthesized using 8 µg poly (A) + rna, avian myeloblastosis virus reverse transcriptase (Life Sciences) and an oligo (dT) primer. The DNA-RNA hybrid was subjected to digestion with 5 RNase H (BRL) and the second strand was synthesized using DNA polymerase I (New England Biolabs). The product was methylated with Eco R1 methylase (New England Biolabs), bluntly ligated to Eco

R1 linkers (New England Biolabs), were digested with Eco RI and size-selected on a low melting point agarose gel. cDNA larger than 500bp was ligated to Xgt10, which had already been subjected to digestion with Eco RI and dephosphorylated (Stratagene). The ligation product was then packed 15 (Stratagene gold).

The umbilical vein endothelial cell and HL-60 cDNA libraries were then plated on 20,000 PFU / 150 mm plate. Recombinant DNA was double transferred to nitrocellulose filters, denatured in 0.5 M NaOH / 1.5 M NaCl, neutralized in 1 M Tris, pH 7.5 / L, 5 M NaCl and baked at 80 ° C for 2 hours (Benton, WD, et al , Science 196; 180-182 (1977)). Filters were prehybridized and hybridized in 5X SSC containing 5X Denhardt's solution, 50 mM NaPO4 and 1 µg / ml salmon sperm DNA. Prehybridization was carried out at 45 ° C for 1 hour.

The hybridization was performed using 32bp (5-TTGGGCTGGTCACAGGAGGTGGAGCAGGTGAC) or 47bp (5 -GAGGTGTTCTCAAACAGCTCCAGGCCCTGGGGCCGCAGGTCCAGCTC) anti-sense oligonucleotides based on, analogous to, R., J. Molec. Blol., 183: 1-12 (1985)). Oligonucleotides were end-labeled with γ- (32P) ATP using T4 polynucleotide kinase and conditioned, recommended by the manufacturer (New England Biolabs). After overnight hybridization, the filters were washed twice with 2X SSC / 0.1% SDS for 30 min. at 45 ° C. Phages were isolated from these plagues, which exhibited hybridization and purified by subsequent re-plating and rescreening.

Cloning of the IGAM-1 gene through the use of anti-ICAM antibody.

Alternatively, the gene for ICAM-1 can be cloned through the use of anti-ICAM-1 antibody. DNA or more preferably cDNA is extracted and purified from a cell capable of expressing ICAM-1. The purified cDNA is subjected to fragmentation (by clipping, endonuclease digestion, etc.) to produce a pool of DNA or cDNA fragments. DNA or cDNA fragments from this pool are then cloned into an expression vector to produce a genomic library of expression vectors, each of which contains a unique cloned DNA or cDNA fragment.

Example 17

Analysis of the cDNA clones.

Phage DNA from positive clones was digested with Eco RI and examined by Southern analysis using a cDNA from a clone as a probe. Maximum size cDNA insertions that cross-hybridized were subcloned into the Eco RI site of plasmid vector pGEM 4Z (Promega). HL-60 subclones containing 30CDNA in both orientations were deleted by exonuclease

Ill digestion (Henikoff, S., Gene 28: 351-359 (1984)) according to the manufacturer's recommendations (Erase-a-Base, Promega). Progressively deleted cDNA was then cloned and subjected to dideoxynucleotide chain termination sequencing (Sanger, F. et al., Proc. Natl ♦

Acad. Sci. (USA) 74: 5463-5467 (1977) according to the manufacturer's recommendations (Sequenase, U.S.A.)

Biochemical). HL-60 cDNA 5 and coding regions were sequentially complemented to both strands and the 3 'region was sequenced approximately. 70% on both strings. A representative endothelial cDNA was sequenced and most of its length by shotgun cloning of 4bp recognition restriction 10 ktlone enzyme fragments.

The cDNA sequence of an HL-60 and an endothelial cell cDNA was established (Fig. 8). The 3023 bp sequence Contains a short 5 non-translated region and a 1.3 kb 3 non-translated region with a consensus polyadenylation signal at position 2966. The longest open reading frame occupies at first ATG 1 position 58 and ends at a TGA terminating triplet at position 1653. Identity between the translated amino acid sequence and sequences determined from eight different tryptic peptides with a total of 91 amino acids (underlined in Figure 8) confirmed that authentic ICAM-1 cDNA clones were been isolated. Based on sequence analysis, there is a loss. 8 shows possible peptides within the ICAM-1 sequence recognized by LFA-1.

25 DK 176020 B1! | 78

Table 8

Amino acid sequences of ICAM-1 tryptic peptides.

Peptide Residues Sequence

1 5 J 14-29 X G S V L VTCSTSCDQPK

U 30- 39 L L G I E T P L <P) (K)

50 78-85 (T) F L T V Y X T

X 89- 95 VELAPLP

AA 161-182 X ELDLRPQ, GLE -

10 L F E X T S A P X Q L

K 232-246 LNPTVTYGXDSFSAK

45 282-295 S F P A P N V (T / I) L X K P Q (V / L) 15 - Indicates that the sequence continues on the next linile. Underlined sequences were used to prepare oligonucleotide probes.

Hydrophobicity analysis (Kyte, J. et al., J.

20 Molec. Blol., 157: 105-132 (1982)) suggests the presence of a 27 residual signal sequence. The designation of the +1 glutamine is consistent with our inability to obtain N-terminal sequence on 3 different ICAM-1 protein preparations; glutamine can cyclize to 25 pyroglutamic acid, resulting in a blocked l-terminus. The translated sequence from 1-453 is substantially hydrophilic, followed by a 24 residual hydrophobic putative transmembrane region. The transmembrane region is followed by several charged residues containing 30 in a 27 residual putative cytoplasmic region.

The predicted size of the polypeptide chain developed is 55,219 daltons, which is in good agreement with the observed size of in DK 176020 B1 79 55,000 for deglycosylated ICAM-1 (Dustin, ML et al., J. Immunol. 137: 245-254 (1986)). Eight N-linked glycylation sites are predicted. Lack of asparagine in the tryptic peptide sequences of two of these sites confirms their glycosylation and their extracellular orientation. Assuming 2,500 daltons per high-mannose N-linked carbohydrate, a size of 75,000 daltons is predicted for the ICAM-1 precursor compared to the observed six of 73,000 daltons (Dustin, ML, et al., J. Immunol. 137: 245 -254 (1986)). After conversion of high mannose to complex carbohydrate, the fully developed ICAM-1 glycoprotein ranges from 76 to 114 kd, depending on cell type 1 (Dustin, ML, et al., J. Immunol. 137: 245-254 (1986)) . Thus, ICAM-1 is a potent glycosylated but otherwise a typical integral membrane protein.

Example 18 ICMA-1 is an integrin binding member of the immunoglobulin 20 supergene family.

Copying of ICAM-1 internal repeats is performed using the "Microgenie protein alignment program" (Queen, C., et al., Nacl. Acid. Res., 12: 581-599 (1984)), followed by inspection. Copying of ICAM-1 to IgM, N-CAM and MAG was performed using the "Microgenie and the ALIGN program" (Day-hoff, MO, et al., Meth. Enzymol. £ 1: 524-545 (1983)) .

Four protein sequence databases stored by the National Biomedical Research Foundation were searched for 30 protein sequence similarities using the FASTP program by Williams and Pearson (Llpman, D.J., et al.,

Science 227: 1435-1439 (1985)).

Since ICAM-1 is a ligand of an integrin, it was not envisaged that it would be a member of the 80og 176020 B1 immunoglobulin supergene family. However, inspection of the ICAM-1-I sequence shows that it meets all the criteria proposed for membership of the immunoglobulin supergene family. These criteria are referred to in turn in the following.

The entire extracellular region of ICAM-1 is constructed from 5 homologous immunoglobulin-like regions shown in FIG. 9A. Areas 1-4 are 88, 97, 99, and 99 residues long, respectively, and are thus characterized by typical Ig area major rice; area 5 is truncated within 68 residues. Searches of the NBRF database using the FASTP program revealed significant homologies between members of the immunoglobulin supergene family, including IgM and igG C regions, ΤΙ 5 cell receptor α-subunit variable region and α-1-β glycoprotein (Fig. 9B). D).

Using the above information, the amino acid sequence of ICAM-1 was compared with amino acid sequences of other members of the immunoglobulin supergene family.

In Three types of Ig superfamily regions, V, Cl and C2 are differentiated. Both V and C regions are constructed from 2 β-plates bound together by the intra-region disulfide bond; v regions contain 9 anti-25 parallel β strands, while C regions contain 7. Constant regions are divided into C1 and C2 sets based on characteristic residues shown in Figs. 9A. The C1 kit comprises proteins involved in antigen recognition. The C2 set comprises several Fc receptors and proteins involved in call adhesion including CD2, LFA-3, MAG and NCAM. ICAM-1 regions were found to be most strongly homologous to regions from the C2-set placing ICAM-1 in this set; this is reflected in stronger similarity to residues at C2 end in C1 regions as shown for the β-strands B-F in Figs. 9. ICAM regions are thus much better aligned with β-strands A and G in C2 regions than with these strands in V and Cl regions, allowing 5 good groupings across the entire C2 region strength. Arrangement of C2 regions from NCAM, MAG and α Ι-β-glyco protein is shown in Figs. 9B and 9C; identity ranges from 28 to 33%. In addition, arrangement of T cell receptor Va, 27% identity and igM C region 3 34% is shown (Figs. 9B, 10.9D) ·

One of the most important characteristics of immunoglobulin regions is the disulfide-linked cysteine bridge bonds between B and F β-strands which stabilize the β-layer sandwich; in ICAM-1, cysteines are conserved in all cases except in strand f of region 4, where a leucine is found which may be directed to the sandwich and stabilize the contact as suggested for certain other B and C2 sets. The distance between the cysteines (43, 50, 52 and 37 residues) is as described for the C2 set.

To test for the presence of intrac chain disulfide bonds in ICAM-1, endothelial cell ICAM-1 was exposed to SDS-PAGE under reducing and non-reducing. conditions. Endothelial cell ICAM-1 was used because it exhibits less glycosylation heterogeneity than for JY or hair cell spleen ICAM-1 and allows greater sensitivity to shifts in Mr. Therefore, ICAM-1 was purified from 16 hours of LPS (5 µg / ml) stimulated umbilical vein endothelial cell cultures by immunoaffinity chromatography as described above. Acetone precipitated ICAM-1 was resuspended in sample buffer (Laemmli, U.K., Nature 227: 680-685 (1970)) with 0.25% 2-mercaptoethanol or 25 mM lodacetamide and brought to 100 ° C for 5 min. The samples were subjected to SDS-PAGE 4670 and silver staining 4613. Endo-82 DK 176020 B1 cell-cell ICAM-1 had a clear Mr of 100 Kd under reducing conditions and 96 Kd under non-reducing conditions, which strongly indicates the presence of intrac chain disulfides in native ICAM-1.

The use of the primary sequence for predicting the secondary structure (Chou, PY, et al., Blochem. 12: 211-245 (1974)} showed the 7 expected β-strands in each ICAM-1 region, labeled ag at the top of Fig. 9A, which exactly satisfies the prediction of a j 10 immunoglobulin region and corresponds to the positions of strands AH in immunoglobulins (bottom of Fig. 9A). Area 5 lacks the A and C strands, but as these form strata, the layers may the layers are still forming, for example, where strand D is replaced with strand C, as suggested for 15 other C2 regions, and the characteristic disulfide bond between the B and F strands would be unaffected.

Thus, the criteria for area size, sequence homology, cyst formation of the presumed intra-area disulfide bond, presence of disulfide bonds, and predicted β-layer structure are all met to include ICAM-1 in the immunoglobulin supergenic family.

ICAM-1 was found to be highly homologous to NCAM and MAG glycoproteins from the C2 set. This is of particular interest as both NCAM and MAG mediate cell-cell adhesion. NCAM is important in neuron-neuron and neuromuscular interactions (Cunningham, BA, et al., Science 236: 799-806 (1987)), while MAG is important in neuron-oligodendrocyte and oligodendrocyte-oligodendrocyte interactions during myelination. (Poltorak, M., et al., J. Cell Biol. 105: 1893-1899 (1987)). Cell surface expression of NCAM and MAG is developmentally regulated during nervous system formation and 83 'DK 176020 B1 myelination, respectively, analogously to the regulated introduction of ICAM-1 by inflammation (Springer, T.A., et al., Ann.

Rev. Immunol. 5; 223-252 (1987)). ICAM-1, NCAM (Cunningham, BA, et al., Science 236: 799-806 (1987)) and MAG (Salzer, JL, et al., J. Cell. Biol. 104: 957-965 (1987) ) are similar in major structure as well as homologous since each is an integral membrane glycoprotein constructed from 5 C2 regions, forming the N-terminal extracellular region, although some additional non-Ig-like sequences exist in NCAM between the last C2-10 region and the transmembrane region. ICAM-1 stands throughout its length, including in transmembrane and cytoplasmic regions, aligned with MAG with 21% identity; the same% identity is found when comparing 5 areas between ICAM-1 and NCAM-1. A schematic comparison between the secondary structure of ICAM-1 and MAG is shown in Fig.10. Area-by-area comparisons show that the homology level between the areas within ICAM-1 and NCAM molecules (x + standard deviation, 21 + 2.8% and 18.6 + 3.8%, respectively) is the same as 20 the homology level when comparing ICAM-1 regions with NCAM and MAG regions (20.4 + 3.7 and 21.9 + 2.7, respectively). Although there is evidence of alternative cleavage in the C-terminal regions of NCAM (Cunningham, B.A., et al., Science 236: 799-806 (1987); Barthels, D., et al.

EMBO J. 6: 907-914 (1987)) and MAG (Lai, C., et al.

Proc. Natl. Acad. Sci. (USA) 84 4377-4341 (1987)), no evidence of this has been found in the sequencing of endothelial or HL-60 ICAM-1 clones or in the studies of the ICAM-1 protein backbone and precursor in a 50 long range of cell types (Dustin ML, et al., J. Immunol. 137: 245-254 (1986)).

ICAM-1 acts as a ligand for LFA-1 in lymphocyte interactions with different cell types. Lympho-84 DK 176020 B1 cytes bind to ICAM-1 incorporated in artificial membrane attachments, and this requires LFA-1 on the lymphocyte, which directly detects LFA-1 interaction with ICAM-1 (Marlin, SD, et al., Cell 51: 813-819 (1987)). LFA-15 is a leukocyte integrin and has no immunoglobulin-like features. Leukocyte integrins comprise one integrin subfamily. The other two subfamilies mediate cell-to-matrix interactions and recognize the sequence RGD within their ligands, which includes fibronetin, vitronecin, collagen and fibrinogen (Hynes, RO, Cell 48: 549-554 (1987); Ruoslahti, E., et al., Science 238: 491-497 (1987)). The leukocyte integrins are only expressed on leukocytes, are involved in cell-cell interactions and the only known ligands are ICAM-1 and iC3b, a fragment of the complementary component C3 which does not exhibit immunoglobulin-like features and is recognized by Mac-1 (Kishimoto, T.K., et al., In: Leukocyte Typing III, McMichael, M. (ed.), Springer-Verlag, New York (1987); Springer, T.A., et al., Ann ♦ Rev. Immunol 5: 20 223-252 (1987); Anderson, DC, et al., Ann. Rev. Med. 38: 175-194 (1987)). Based on sequence analysis, possible peptides within the ICAM-1 sequence, recognized by LFA-1, are shown in Table 9.

DK 176020 B1 85

Table 9

Peptides within the ICAM-1 sequence, which may be recognized by LFA-1.

5 -LRGEKEL- -RGEKELKREP- -LRGEKELKREPAVGEPAE- -PRGGS- -PGNNRK- 10 -QEDSQPM- -TPERVELAPLPS- -RRDHHGANFS- -DLRPQGLE- ICAM-1 is the first example of a member of the immunoglobulin family of genes, . Although both of these families play an important role in cell adhesion, interactions between them have not been expected in the past. In contrast, interactions within the immunoglobulin gene superfamily are quite common. It is very likely that further examples of interactions between the integrin and immunoglobulin families will be found. LFA-1 recognizes a ligand, different from ICAM-1 (Springer, T.A., et al., Ann. Rev. Immunol. 5: 223-252 (1987)), and the leukocyte integrin Mac-1 recognizes a ligand, for; different from C3bi in neutrophil-neutrophil adhesion (Anderson, D.C., et al., Ann. Rev. Med. 36: 175-194 (1987)). Purified MAG-containing vesicles further bind to neurites which are MAG, and thus MAG must be capable of heterophilic interaction with a distinct receptor (Poltorak, M., et al., J. Cell Biol. 105: 1893- 1899 (1987)).

The role of NCAM in neural-neural and neural-muscular cell interactions has been suggested to be due to the homosexual NCAM-NCAM interactions (Cunningham, B.A., et al., Science 236: 799-806 (1987)). The important role of MAG in interactions between adjacent, revolving loops in Schwann cells surrounding axons during myelin sheath formation may be due to interaction with a distinct receptor or homosexual MAG-MAG interactions. The homology between NCAM and the frequent occurrence of area-domain interactions within the immunogobulin supergenic family raises the possibility that ICAM-1 could be involved in homophobic interactions as well as ICAM-1-LFA-1 heterophilic interactions. However, binding of B-lymphoblast cells I co-expressing similar densities of LFA-1 and ICAM-1j to ICAM-1 in artificial or cellular monolayers can, however, be completely inhibited by pretreatment of the B-lymphoblast with LFA-1-MAb , whereas adherence is unaffected by B lymphoblast pretreatment with ICAM-1-MAb. Pre-treatment of the monolayer with ICAM-1-Mab abolishes complete binding (Dustin, M.L., et al., J. Immunol.

] J7: 245-254 (1986); Marlin, S.D. et al., Cell 51: 813-819 (1987)). These findings indicate that if ICAM-1 homosexual interactions occur at all, they must be much weaker than heterosexual interactions with LFA-1.

The possibility that the leukocyte integrins know ligands in a fundamentally different way is consistent with the presence of a 180 residue sequence in their α-subunits that may be important in ligand binding and not found in the RGD recognition integrins (Corbi, A., et al. (EMBO J. 6: 4023-4028 (1987)) Although it has been suggested that Mac-1 recognizes RGD sequence found in iC3b 5086, there is no RGD sequence in ICAM-1 (Fig. 8). .

87 DK 176020 B1

This is consistent with the inability of the fibronetic peptide GRGDSP and the control peptide GRGESP to inhibit ICAM-1-LPA-1 adhesion (Marlin, S.D., et al., Cell 51: 813-819 (1987)). However, related sequences, such as PRGGS and RGEKE, are found in ICAM-1 in regions which are said to form a loop between β-strands a and b in regions 2 and c and d, respectively, in region 2 (Figs.

9) and should thus have the opportunity for recognition.

It is of interest that the homologous MAG molecule contains an RGD sequence between regions 1 and 2 (Poltorak, M., et al., J. Cell Biol. 105: 1893-1899 (1987); Salzer, J.L. , et al., J. Cell, Biol. 104: 957965 (1987)).

Example 19 Southern and Northern Blots.

Southern blots were performed using 5 µg of genomic DNA extracted from three cell lines; BL2, t a Burkitt lymphoma cell line (a gift from Dr. Gilbert Lenoir); JY and Er-LCL, EBV-transformed B-lympho-blastoid cell lines.

DNA was subjected to digestion with 5X of the amount of Bam H1 and Eco RI endo nucleases (New England Biolabs) recommended by the manufacturer. After electrophoresis through a 0.8% agarose gel, DNA was transferred to a nylon 25 membrane (Zeta Probe, BioRad). The filter was prehybridized and hybridized, following the standard procedures using ICAM cDNA from HL-60 labeled x with α- (32P) d XTP's at random priming (Boehringer Mannheim). Northern blots were performed using 30 of 20 µg total RNA or 6 µg poly (A) + RNA. RNA was denatured and electrophoresed through a 1% agarose-formaldehyde gel and electrophoresed to Zeta probe. Filters were prehybridized and hybridized as described previously (Staunton, D.E., et al., Embo J.

6_: 3695-3701 (1987)) using the HL-60 cDNA probe of 3β-labeled oligonudeotide probes (described above).

Southern blots using the 3 kb cdna probe and genomic DNA subjected to digestion with Bam H1 and Eco Ri showed simple dominant hybridization pigments of 20 and 8 kb, respectively, suggesting a single gene and most of the coding information is found within 8 kb. In blots with three different cell lines, there was no evidence of restriction fragment polymorphism.

Example 20 Expression of the ICAM-1 gene.

An "expression vector" is a vector which (due to the presence of appropriate transcription and / or translation control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into a vector and thereby producing a polypeptide or protein. Expression of cloned sequences occurs when the expression vector is introduced into a suitable host cell. Thus, if a prokaryotic expression vector is used, the appropriate host cell will contain any prokaryotic cell capable of expressing the cloned sequences. Similarly, if a eukaryotic expression vector is used, the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences.

Since eukaryotic DNA may contain intervening sequences and such sequences cannot be properly produced in prokaryotic cells, it is preferred to use cDNA from a cell capable of expressing a prokaryotic genomic gene. ekspressionsvektorbibliotek. Methods for producing cDNA and preparing a genomic library are disclosed by Maniatis, T., et al. (Molecular Cloning: 5 A Laboratory Manual, Cold Spring Harbor Press,

Cold Spring Harbor, NY (1982)).

The genomic library of the aforementioned expression vector is used to generate a bank of host cells (each containing a member of the library).

The expression vector can be introduced into the host cell by a variety of means (i.e., transformation, transfection, protoplast fusion, electroporation, etc.). The expression vector-containing cell bank is clonically propagated and its members are tested individually (using an immunoassay) to determine whether they produce a protein capable of binding to antiICAM-1 antibody.

The expression vectors of the cells producing a protein capable of binding to anti-ICAM antibody are then further analyzed to determine if they express (and thus contain) the entire ICAM-1 gene if they express only (and contains) a fragment of the ICAM-1 gene, or whether they express (and contain) a gene whose product is not ICAM-1, although immunologically related to ICAM-1. Although such an assay can be performed with any suitable agent, it is preferred to determine the nucleotide sequence of the DNA or cDNA fragment cloned into the expression vector. Such nucleotide sequences are then examined to determine if they are capable of encoding polypeptides that have the same amino acid sequence as ICAM-1's tryptic digestion fragments (Table 5).

90 DK 176020 B1

Thus, an expression vector containing a DNA or cDNA molecule encoding the ICAM-1 gene can be recognized by: (i) The ability to directly express a protein as capable of binding to anti-5 * ICAM -1-antibody; and (ii) the presence of a nucleotide sequence capable of encoding each of the tryptic fragments of ICAM-1. The cloned DNA molecule of such an expression vector can be removed from the expression vector and isolated in pure form.

10

Example 21

Functional activities of purified ICAM-1.

In cells, ICAM-1 usually acts as a surface protein attached to the cell membrane. Therefore, after the molecule was reconstituted, the function of purified ICAM-1 was tested in artificial lipid membranes (liposomes or vesicles) by dissolving the protein in detergent-solubilized lipids, followed by removal of the detergent by dialysis. ICAM-1 purified from JY cells and eluted in detergent octyl glucoside, as described above, was reconstituted in vesicles, and ICAM-1-containing vesicles were fused to SD glass or plastic culture wells allowing the detection of cell binding to the protein.

7 5 Manufacture of planar membranes and plastic-bound vesicles.

Vesicles were prepared by the method of Gay et al. (J. Immunol. 136: 2026 (1986)). Briefly, egg phosphatidylcholine and cholesterol were dissolved in trichloromethane and mixed in a molar ratio of 7: 2. The lipid mixture was dried to a thin film under rotation under a stream of nitrogen gas and then lyophilized for 1 hour to remove all traces of trichloromethane. The lipid film was then dissolved in 1% ethyl glucoside / 0.14 M Nacl / 20 mM Tris (pH 7.2) to a final concentration of phosphatidylcholine of 0.1 mM. Ca.

5 µg of purified ICAM-1 or human glycophorin (Sigma Chemical Co., St. Louis, MO) as a control membrane glycoprotein was added to each of the dissolved lipids. The protein-lipid detergent solution was then dialyzed at 4 ° C against 3 holdings of 200 volumes of 20 mM 10 Tris / 0.14 M NaCl, pH 7.2, and a hold of HBSS.

Plane membranes were prepared by the method of Brian et al., Proc. Watl. Acad. Sci. 81: 6159 (1984). Cover coverslips (with a diameter of 11 mm) for 15 minutes. in a 1: 6 dilution of 7X detergent (Linbro), washed overnight in distilled water, left immersed in 70% ethanol and air dried. An 80 μΐ drop of a suspension containing either ICAM-1 or glycop-i horln was placed on the bottom of the wells of a 24-well cluster test plate and the prepared coverslips flowed easily on the top 20. After 20-30 min. incubation at room temperature, the wells were filled with HBSS and the coverslip was inverted to bring the planar membrane up. The wells were then washed thoroughly with HBSS to remove unbound vesicles. The flat membrane surface was never exposed to 25 air.

During the experiments with flat membranes fused to glass surfaces, it was found that vesicles containing ICAM-1 bind directly to the plastic surface of multi-well tissue culture plates and retain functional effect 30 as evidenced by specific cell binding. Such vesicles are then referred to as "plastic-bound vesicles" (PBV), since the nature of lipid vesicles bound to plastics is not determined. Plastic-bound vesicles were prepared by adding 30 μΐ vesicle suspension directly to the bottom of wells in 96-well tissue culture trays (Falcon) followed by incubation and washing as described for planar membranes.

5

Celleadhæsionsprøver.

Cell adhesion tests using planar membranes or plastic-bonded vesicles were both conducted in substantially the same way, except that the cell number and volumes of PBV samples were reduced to one-fifth of that used in planar membrane samples.

Normal lymphocytes from normal controls and a patient with leukocyte adhesion defect (LAD) whose 15 cells do not express LFA-1 (Anderson, DC et al., J. Infect. Dis. 152: 668 (1985)) were prepared by culturing peripheral mononuclear blood cells at 1 µg / ml Concanavalin-A (Con-A) in RPMI-1640 plus 20% FCS at 5 x 10 5 cells / ml for 3 days. The cells were then washed twice with RPMI and once with 5 mM methyl-α-D-mannopyranoside to remove residual lectin from the cell surface. The cells were grown in RPMI / 20% FCS containing 1 ng / ml recombinant IL-2 and used between 10 and 22 days after culture initiation.

For detection of cell binding to planar membranes or PBV, Con-A blasts, T-lymphoma SKW-3 and EBV-transformed B lymphoblastoid cell lines JY (LFA-1 positive) and LFA-1 defective lymphoblastoid cell line (BBN) were radiolabelled. ) (derived from patient 30 # 1, Springer, TA et al., J. Exper. Med. 160; 1901-1918 (1986)) by incubating 1 x 10 7 cells in 1 ml of RPMI-1640/10% FCS. with 100 µCi of Na51 CrO4 for 1 hour at 37 ° C, followed by 4 washes with RPMI-1640 to 93 DK 176020 B1 removal of Unincorporated marks. In monoclonal antibody blocking experiments, cells or plastic-bound vesicles were pretreated with 20 µg / ml purified antibody 1 RPMI-1640/10% FCS at 4 ° C for 30 min, followed by 54 washes to remove unbound antibody. In experiments for the effect of divalent cations on cell binding, cells were once vaccinated with Ca 2+, Mg 2+ -free HBSS plus 10% dialyzed FCS, and CaCl and MgCl were added to the indicated concentrations. In all experiments, cells and planar membranes or PBV were pre-balanced at the appropriate temperature (4 ° C, 22 ° C or 37 ° C) in the appropriate sample buffer.

To measure cell binding to purified ICAM-1, 51 Cr labels were centrifuged (5 x 10 5 EBV-15 transformants 1 flat membrane samples; 1 x 10 5 EBV transformants or SKW-3 cells, 2 x 10 5 Con-A blisters in PBV-samples). for 2 min. at 25 x g on planar membranes or PBV, followed by incubation at 4 ° C, 22 ° C or 37 ° C for 1 hour. After incubation, 20 unbound cells were removed by eight cycles of loading and aspiration with buffer preset to the appropriate temperature. Bound cells were determined by solubilizing the well content with 0.1 N NaOH / 1% Triton x-100 and counting on a y counter. Percent cell binding was determined by dividing cpm from bound cells with input cell-associated cpm. In 1 plane membrane samples, the input cpm was corrected for the ratio of the surface area of the coverslip to the surface area of the culture wells.

In these samples, EBV-transformed B-30 lymphoblastoid cells, SKW-3 T lymphoma cells, and Con-A T lymphoblasts bound specifically to ICAM-1 in artificial membranes (Figs. 11 and 12). The binding was specific since the cells bound very weakly to control planar membranes or vesicles containing equivalent amounts of another human cell surface glycoprotein, glycophorline. Further, LFA-1-positive EBV transformants and Con-A blasted while their LFA-1-negative counterparts did not bind 1 to any significant extent, indicating that binding was dependent on the presence of LFA-1 on the cells.

Both the specificity of cell binding and dependence on cellular LFA-1 were confirmed in monoclonal antibody-10 blocking experiments (Fig. 13). The binding of JY cells could be inhibited by 97% when ICAM-1 containing PBV was pretreated with anti-ICAM-1 monoclonal antibody RR1 / 1. Pre-treatment of the cells with the same antibody had little effect. Conversely, the anti-LFA-1 monoclonal antibody TS1 / 18 binding was inhibited by 96%, but only when the cells, but not the PBV, were pretreated.

A control antibody T $ 2/9 reactive with LFA-3 (another lymphocyte surface antigen) had no significant inhibitory effect, neither when cells nor PBV were pretreated. This experiment shows that ICAM-1 itself, and not certain less contaminating materials in the artificial membranes, mediates the observed cellular adhesion and that the adhesion is dependent on LFA-1 on the binding cell.

Furthermore, the binding of cells to ICAM-1 in artificial membranes shows two other properties of the LFA-1 dependent adhesion system: temperature dependent and the necessity of divalent cations. As shown in FIG. 14 bonded Con-A blasts to ICAM-1 in PBV most effectively at 30 ° C, partially at 22 ° C and very weakly weighed 4 ° C. As shown in FIG. 15, the bond was completely dependent on the presence of divalent cations. At physiological concentrations, Mg2 + alone was sufficient for maximal 95 'DK 176020 B1 template cell binding, whereas Ca2 + alone provides very low levels of binding. However, one-tenth of Mg 2 '1' of normal concentration together with Ca 2+ was synergistic and produced maximum binding.

In summary, the specificity of cell binding to purified ICAM-1, incorporated in artificial membranes, shows the specific inhibition with monoclonal antibodies, and the temperature and divalent cation requirement show that ICAM-1 is a specific ligand for the LFA-1 dependent adhesion system.

Example 22

Expression of ICAM-1 and HLA-DR 1 allergic and toxic laptest reactons.

Fifteen skin biopsies from five normal subjects were searched for their expression of ICAM-1 and HLA-DR. It was found that while the endothelial cells of some blood vessels usually expressed ICAM-1, no ICAM-1 was expressed on normal skin keratinocytes. No staining for HLA-DR on some keratinocytes from normal skin biopsies was observed. The kinetics of expression of ICAM-1 and class II antigens were then examined on cells in biopsies from allergic and toxic skin lesions. It was found that half of the six individuals studied had keratinocytes expressing ICAM-1 four hours after the use of hapten (Table 10). There was an increase in the percentage of individuals expressing ICAM-1 on their keratinocytes with time to expose the hapten, as well as an increase in the intensity of staining, indicating more ICAM-1 expression per keratinocyte up to 48 hours. In fact, at this time, some keratinocytes in all biopsies are stained positive for ICAM-1. At .72 hours (24 95 DK 176020 B1 for hours after the hapten was removed), 7 of 8 individuals still had ICAM-1 expressed on their keratinocytes, while the expression of ICAM-1 on an individual migrated between 48 and 72 hours.

DK 176020 B1 97

Table 10

Kinetics of introduction of ICAM-1 and HLA-DR on kerati-I nocytes from allergic lab test biopsies.

5 _

Time after the number of ICAM-1 HLA-DR ICAM-1 and patch application of HLA-DR alone

(H) _ _blopsier; ____

Normal skin_5_0_0_0_ 10 Allergic patch test 4 6 3a 0 0! 8 9 3 0 0 24 8 7 0 0 15 48b 8 5 0 3 72 8 6 0 .1 20 25 a Samples were considered positive if at least small clusters of keratinocytes were stained. b All patches were removed at this time.

in DK 176020 B1 98

Histologically, the staining pattern of ICAM-1 on keratinocytes from blopsis was taken four hours after hapten application as usual in small clusters. At 48 hours, ICAM-1 was expressed on the surface of the majority of the 5 keratinocytes, seeing no difference between the center of the injury and the periphery, the intensity of staining decreasing as the keratinocytes approached the stratum corneum. This was found in biopsies taken from the center and periphery of the lesions. At this time j 10, the lap test was also positive (infiltration, erythema and vesicles). No difference 1 ICAM-1 expression was observed when different haptens were applied to sensitive individuals. In addition to the keratinocytes, ICAM-1 was also expressed on some on-site mononuclear cells and endothelial cells with the damage in addition to the keratinocytes.

The expression of HLA-DR on keratinocytes on the allergic skin lesions was less frequent than the expression of ICAM-1. None of the subjects studied had keratinocyte lesions that stained positively for 20 HLA-DR up to 24 hours after hapten application. In fact, only four biopsy specimens had keratinocytes expressing HLA-DR and no biosies had keratinocytes positive for HLA-DR and not ICAM-1 (Table 10).

In contrast to the allergic lap test injury, the toxic lap test injury induced by croton oil or sodium lauryl sulfate had keratinocytes that showed little, if any, ICAM-1 on their surfaces at all times tested (Table 11). In fact, only 30 of the 14 subjects exposed to the toxic laptest keratinocytes expressing ICAM-1 in their injury 48 hours after patch application, which is the optimal time for ICAM-1 expression allergic laptest individuals. In addition, unlike the allergic lap test biopsies, HLA-DR was not expressed on keratinocytes of toxic lap test damage.

These data indicate that ICAM-1 is expressed in immune-based inflammation and not in toxic-based inflammation, and thus the expression of ICAM-1 can be used to distinguish between immunobased- and toxic-based inflammation, such as acute renal failure in renal transplant patients, where is difficult to determine whether the failure is due to rejection or nephrotoxicity of the immunosuppressive therapeutic agent. Renal biopsy and assessment of up-regulation of ICAM-1 expression will allow differentiation between 15 immune-based rejection and non-immune-based toxicity response.

* DK 176020 B1 100

Table II

Kinetics of introduction of ICAM-1 and HLA-DR on keratinocytes from toxic laptest biopsies.

5 _

Time after Number of ICAM-1 HLA-DR ICAM-1 and

patch application of HLA-DR alone

(hours) _blopsler_ 4 4 0 0 0 8 3 la 0 0 10 24 3 1 0 0 48b 14 10 0 72 3 1 0 0 a Samples were considered positive if at least 15 small clusters of keratinocytes were stained.

b All patches were removed at this time._ DK 176020 B1 101

Example 23

Expression of ICAM-1 and HIA-PR in benign cutaneous diseases'

Cells from skin biopsies with lesions from patients 5 with different types of inflammatory skin diseases were examined for their expression of ICAM-1 and HLA-DB.

Some keratinocytes 1 biopsies with allergic contact eczema, pemphigoid / pemphigus and lichen planus expressed ICAM. Lichen planus biopsies showed the most intense staining with a pattern similar to or even stronger than those found in the 48-hour allergic lap test biopsies (Table 12). In accordance with the results seen in the allergic lap test, the most intense iCAM-1 staining was observed in 15 sites with vigorous mononuclear cell infiltration. Eight of the 11 lichen planus biopsies tested were further positive for HLA-DR expression on keratinocytes.

The expression of ICAM-1 on keratinocytes from skin biopsies from patients with exanthema and urticaria 20 was less pronounced. Only four of the seven patients tested with these diseases had keratinocytes expressing ICAM-1 at the lesion site. HLA-DR expression appeared in only one patient, and this was in conjunction with ICAM-1.

25 Endothelial cells and part of the mononuclear cell infiltrates from all the benign inflammatory cells tested. rich skin diseases expressed ICAM-1 to varying degrees.

DK 176020 B1 102

Table 12

Expression of ICAM-1 and HLA-DR on keratinocytes from benign cutaneous diseases.

5 Diagnosis The number of ICAM-1 HLA-DR ICAM-1 and of cases alone HLA-DR_

Allergic Contact Eczema 5 3a 0 2

Lichen Planus 11 30 θ 10 Pemhigoid /

Pemhigus 2,200

Exanthema 3 2 0 0

Urticaria 4 1 01 15 »i Λ

Samples were considered positive if at least 20 small clusters of keratinocytes were stained.

i i i i DK 176020 B1 103

Example 24

Expression of ICAM-1 on keratinocytes from skin lesions associated with psoriasis.

The expression of ICAM-1 in skin biopsies from 5 pa-5 patients with psoriasis was examined before initiation and periodically during a course of PUVA treatment. Biopsies were obtained from five patients with classic psoriasis confirmed by histology. Biopsies were taken in order before and during the indicated time for PUVA treatment.

PUVA was applied 3-4 times a week. Biopsies were taken 10 from the periphery of psoriasis plagues in five patients and additional biopsies were taken from clinically normal skin from four of the patients.

Fresh skin biopsy pieces were frozen and stored in liquid nitrogen. Six µm cryostat sections were air-dried overnight at room temperature, fixed in acetone for 10 min. and either dyed immediately or wrapped in aluminum foil and stored at -B0 ° C in-I for staining.

Staining was carried out in the following manner.

20 Sections were incubated with monoclonal antibodies and stained by a 3-step immunoperoxidase method (Stein, H., et al., Adv. Cancer Res 42: 67-147, (1984)) using diaminobenzidine H2O2 substrate. Tonsils and lymph nodes were used as a positive control for 25 anti-ICAM-1 and HLA-DR staining. Tissues stained in the absence of a primary antibody were negative controls.

The monoclonal antibodies against HLA-DR were obtained from Becton Dickinson (Mountainview, California). The monoclonal anti-ICAM-1 antibody was R6-5-D6. Peroxida-30 se-conjugated rabbit anti-mouse Ig and peroxidase-conjugated porcine anti-rabbit Ig were acquired from DAKAPATTS, Copenhagen, Denmark. Diaminobenzidine tetrahydrochloride was obtained from Sigma (St. Louis, Mo.).

104 - DK 176020 B1

The results of the study showed that in some blood vessels the endothelial cells express both ICAM-1 1 diseased and normal skin, but the intensity of staining and the number of blood vessels expressing ICAM-1 increased in the psoriasis-5 skin lesions. The pattern of expression of ICAM-1 further varied in keratinocytes from untreated psoriasis skin lesions from the five patients, from only small clusters of cell staining to many stained keratinocytes.

During the course of PUVA treatment, ICAM-1-Ex-10 showed depression in two of the patients (patients # 2 and 3) with widespread reduction that preceded or was consistent with clinical remission (Table 13). Patients Nos. 1, 4, and 5 had diminished and increased ICAM-1 expression winning the PUVA treatment, which correlates to clinical remissions and relapses, respectively. There was no ICAM-1 expression on normal skin keratinocytes before or after PUVA treatment. This indicates that PUVA does not induce ICAM-1 on normal skin keratinocytes.

The observation that the density of the mononuclear cell infiltrate correlated with the amount of ICAM-1 expression on keratinocytes was significant. This relates to both a decreasing number of mononuclear cells in damage during PUVA treatment when ICAM-1 expression also migrated, and an increased number of mononuclear cells during PUVA treatment when ICAM-1 expression on keratinocytes was more prominent. In addition, endothelial cells and dermal mononuclear cells are ICAM-1 positive.

In clinically normal skin, ICAM-1 expression was restricted to endothelial cells without labeling keratinocytes.

The expression of HLA-DR on keratinocytes was variable. There was no HLA-DR positive bloody, nor ICAM-1 positive.

105 DK 176020 B1

In summary, these results show that prior to treatment, ICAM-1 expression is pronounced on the keratinocytes and correlates to a dense mononuclear cell infiltrate.

During PUVA treatment, a pronounced decrease in 5 ICAM-1 staining is seen in parallel with the clinical improvement. Histologically, the dermal infiltrate also decreased. When a clinical relapse was evident during treatment, the expression of ICAM-1 on the keratinocytes and the density of dermal infiltrate increased. When a clinical remission 10 was observed during treatment, there was a concomitant decrease in ICAM-1 staining on the keratinocytes and a decrease in the dermal infiltrate. Thus, the expression of ICAM-1 on keratinocytes corresponds to the density of mononuclear cell infiltrate on the dermis. These data show that clinical response to PUVA dressing results in a pronounced decrease in ICAM-1 expression on keratinocytes in parallel with a more moderate decrease of the mononuclear cells. This indicates that ICAM-1 expression on keratinocytes is responsible for initiating and maintaining the dermal infiltrate and that PUVA treatment down-regulates ICAM-1, which in turn decreases the dermal infiltrate and inflammatory response.

In addition, the data indicate that there was a variable HLA-DR expression on keratinocytes during PUVA treatment.

The expression of ICAM-1 on keratinocytes with psoriasis damage correlates with the clinical severity of the injury as well as the size of the dermal infiltrate. Thus, ICAM-1 plays a key role in psoriasis and inhibition of its expression and / or inhibition of its interaction with the CD 18 complex on mononuclear cells will be an effective treatment of the disease. The registration of ICAM-1 expression on keratinocytes will furthermore be an effective tool for diagnosis and prognosis, as well as evaluation of the course of therapy for psoriasis.

in 107 DK 176020 B1

Table 13

Sequential ICAM-1 expression of keratinocytes 1 skin lesions with psoriasis (PS) and clinically normal skin (N) before and during PUVA treatment.

Patient no.

Time before and 1 2 3 4 5 below

PUVA treatment PS PS N PS N PS N PS N

10 0 ++ - ++ - ++ · ♦ ++ -1 day + 1 week ++ - - ++ - +. I ® 2 weeks ++ 3 weeks -H- * 0 15 4 weeks ++ + - ++ * * 5-6 weeks' - ~ 0 7 weeks (++) (+) +++ "* - * 10 weeks (+) 20 _ +++ Many positive keratinocytes.

++ Some positive keratinocytes.

+ Focally positive keratinocytes.

(+) Very few, scattered positive keratinocytes.

30 - no positive staining, x Clinical remission, o Clinical relapse.

DK 176020 B1 108

Example 25

Expression of ICAM-1 and HLA-DR in malignant cutaneous diseases.

Unlike lesions from benign cutaneous conditions 5, the expression of ICAM-1 on keratinocytes from malignant skin lesions was much more variable (Table 14). Of the 23 cutaneous T-cell lymphomas examined, ICAM-positive keratinocytes were identified in only 14 cases. There was a tendency for keratinocytes from 10 mycosis fungoid lesions to lose their ICAM-1 expression upon progression of the disease to more advanced stages. However, ICAM-1 expression was observed on a varied portion of the mononuclear cell infiltrate from most of the cutaneous T cell lymphoma lesions. Among the remaining 15 lymphomas examined, four out of eight had keratinocytes expressing ICAM-1. Of the 29 patients f with malignant cutaneous diseases examined, 5 had keratinocytes expressing HLA-DR without expressing ICAM-1 (Table 14).

t. DK 176020 B1 109

Table 14

Expression of ICAM-1 and HLA-DR on keratinocytes from malignant cutaneous diseases.

5

Diagnosis Number of ICAM-1 HLA-DR ICAM-1 and

_ case only HLA-DR alone

CTCL, MFI 8 la 0 4 CTCL, MFII-III 10 1 2 5 10 CTCL, SS 3 10 2 CTCL, Large cell 2020 CBCL 2 0 0 1

Leukemia Cutis 3111

Histiocytosis XI 00 0 15 _ in α Samples were considered positive if small clusters of keratinocytes were stained.

p.

110 DK 17602Θ B1

Example 26

Effect of anti-ICAM-1 antibodies on the human peripheral mononuclear blood cell proliferation site.

Human peripheral blood mononuclear cells are induced to proliferate in the presence and recognition of antigens or mitogens. Certain molecules, such as the mitogenic concanavalin A, or the T cell | binding antibody OKT3 causes a nonspecific I proliferation of peripheral blood mononuclear cells.

! Human peripheral blood mononuclear cells are heterogeneous in that they consist of subpopulations of cells capable of recognizing specific antigens. When a peripheral blood mononuclear cell capable of recognizing a particular specific antigen meets this antigen, the proliferation of this subpopulation of mononuclear cell is induced. Tetanus toxoid and keyhole limpet hemocyanin are examples of antigens that are recognized by subpopulations of peripheral blood mononuclear cells, but which are not recognized by all peripheral mononuclear cells in sensitized individuals.

The ability of anti-ICAM-1 momoclonal antibody R6-5-D6 to inhibit proliferative responses from human peripheral blood mononuclear cells in systems known to require cell-cell adhesions was tested.

Peripheral blood mononuclear cells were purified on Ficoll-Paque (Pharmacia) gradients according to the manufacturer's instructions. After collection of the interface, cells were washed three times with RPMI 1640 medium and grown in 30 flat-bottomed, 96-well microtiter plates at a concentration of 106 cells / ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine and gentamicin (50 µg / ml).

DK 176020 B1 111 i i

Antigen, either the T cell mitogen, concanavalin A (0.25 µg / ml), the T cell binding antibody, OKT3 (0.001 µg / ml), keyhole limpet hemocyanin (10 g / ml) or tetanus toxoid (1 : 100 dilution from source) was added to cells grown as described above either in the presence or absence of anti-ICAM antibody (R6-5-D6; final concentration of 5 g / ml). days (concanavalin A trial), 2.5 days (OKT3 trial) or 5.5 days ("keyhole limpet" hemocyanin 10 and tetanus toxoid trial) before the tests were completed.

18 hours before the end of the sample, 2.5 µCi 3 H-thymidine was added to the cultures. Cellular proliferation was tested by measuring the incorporation of thymidine into DNA by the peripheral blood mononuclear cells, incorporated thymidine was collected and counted in a liquid scintillation counter (Merluzzi et al., J. Immunol.

139: 166-168 (1978)). The results of these experiments are shown in FIG. 16 (concanavalin A trial), fig. 17 (OCT3 trial), fig. 18 ("keyhole limpet" hemocyanin test) 20 and FIG. 19 (tetanus toxoid trial).

It was found that anti-ICAM-1 antibody inhibits proliferative responses to the nonspecific T cell mitogen, ConA; the non-specific T cell-associated antigen, OKT-3; and the specific anti-25 genes, "keyhole limpet" hemocyanin and tetanus toxoid, in mononuclear cells. The inhibition of anti-ICAM-1 antibody was comparable to the anti-LFA-i antibody suggesting that ICAM-1 is a functional ligand of ICAM-1 and that antagonism of ICAM-1 will inhibit specific get defense system reactions.

DK 176020 B1 112

Example 27 effect of anti-ICAM-1 antibody on the mixed lymphocyte reaction.

As discussed above, ICAM-1 is required for effective cellular interactions during an immune response mediated through LFA-1-dependent cell adhesion. The introduction of ICAM-1 during immune reactions or inflammatory disease allows the interaction of leukocytes with each other and with entodel cells.

* 10 When lymphocytes from two unrelated individuals are grown in each other's presence, blast transformation and cell proliferation of the lymphocytes are observed. This reaction of one population of lymphocytes to the presence of another population of lymphocytes is known as a mixed lymphocyte reaction (MLR), and it is analogous to the reaction of lymphocytes to the addition of mitogens (Immunology The Science of Self-Nonself Discrimination, Klein, j., John Wiley & Sons, NY (1982), pp. 453-4581).

Experiments were conducted to determine the effect of anti-ICAM-1 monoclonal antibodies on human MLR. These experiments were carried out as follows. Peripheral blood was collected from normal healthy donor at venous puncture. The blood was collected in heparinized tubes and diluted 1: 1 at room temperature with Puck's G (GIBCO) Balanced Saline (BSS). The blood mixture (20 ml) was placed on top of 15 ml of a Ficoll / Hypaque density gradient (Pharmacia, density = 1,078 room temperature) and centrifuged at 1000 x g for 20 min. The interface was collected and washed 3X in Puck's G. The cells were counted on a hemacytometer and resuspended in RPMI-1640 culture medium (GIBCO) containing 0.5% gentamicin, 1 mM Lglutamine (GIBCO) and 5% heat inactivated (56 ° C, 30 113 DK 176020 B1 min) human AB sera (Flow Laboratories) (hereafter referred to as RPMI culture medium).

Mouse anti-ICAM-1 (R6-5-D6) was used in these experiments. All monoclonal antibodies (prepared from 5 ascites by Jackson immunoResearch Laboratories, Boston, MA) were used as purified IgGs.

Peripheral blood mononuclear cells (PBMC) were grown in 6.25 x 10 3 cells / ml medium in Linbro round-bottom microtiter plates (# 76-013-05). Stimulator cells from a separate donor were irradiated at 1000 R and cultured with the responder cells at the same concentration. The total volume per culture was 0.2 ml. Controls included responder cells alone and stimulator cells alone. The culture plates were incubated at 37 ° C in a 5% CO 2 humidified air atmosphere for 5 days. The wells were pulsed with 0.5 µCi tri ..... thymidine (3HT) (New Eng land Nuclear) for at least 18 hours per culture. In some cases, a 2-way MLR was performed. The protocol was the same except that the other donor cells I 20 were not inactivated by irradiation.

The cells were harvested on fiberglass filters using an automated multi-sample harvesting apparatus (Skatron, Norway), purifying with water and methanol. The filters were oven dried and counted in Aquasol in a Bec-25 kman (LS-3801) liquid scintillator counter. The results are given as the mean of CPM + mean error for 6 individual cultures.

Loss. Figure 16 shows that purified anti-ICAM-1 monoclonal antibodies inhibit MLR in a dose-dependent manner with significant suppression visible at 20 ng / ml. Purified mouse IgG had little or no suppressive effect. Suppression of MLR by the anti-ICAM-1 monoclonal antibody occurs when the antibody is added within the first 24 hours of culture (Table 17).

DK 176020 B1 114

Table 16

Effect of anti-ICAM-1 antibody on the one-way lymphocyte reaction.

Response counter3 'stimulator counter' Antibody0 iff · Incorporation (CM) 445d ± 143 + - 148 in 17! + - - 698 + 72 10_____ + + 42,626 in 1,579

. - I

+ + nilgG (10.0 µg) 36.882 ± 1.823 (14%) + + mlgG (0.4 µg) 35,500 in 1,383 (17%) 15 + + mlgG (0.02 µg) 42.815 + 1.224 (0%) in +. + R6-5 -D6 (10.0.ug).-8.250 ± 520 (81%) + + R6-5-D6 (0.4 µg) 16.142 ± 858 (62%) + + R6-5-D6 (0.03 µg) 28.844 ± 1.780 (32 µg) %) 20 a. Responder cells (6.25 X 105 / ml) b. Stimulator cells (6.25 X 105 / ml, irradiated at 10000R).

c. Purified monoclonal antibody to ICAM-1 (R6-5-D6) or purified mouse IgG (mlgG) at final concentrations (pg / ml).

cl. Mean + mean error of 5-6 cultures, the number in brackets indicates percent inhibition __ of MLR .__ DK 176020 B1! 115

Table 17

Addition time of anti-ICAM-1.

5 Ra Sb Additions0 3HT Incorporation (CPM)

Medium or antibody cut-off time - - - ♦ ♦ - - * - "* *

Day 0 Oag 1 Day 2 10 - - Medium 205di 14 476 i 132 247 t 75 + medium 189 ± 16 nd nd + - medium 1,860 i 615 nd nd + + medium 41,063 i 2,940 45,955 i 2,947 50,943 i 3,072 ++ R6-5 -D6 17,781 ± 1,293 38,409 ± 1,681 47,308 ± 2,089 (57%) (16%) (7%) a. Resonder cells (, 25 x 10 5 / ml).

20 b. Stimulator cells (6.25 x 10 5 / ml, irradiated at 1000 R).

c. Culture medium or purified monoclonal antibody to ICAM-1 (R6-5-D6) at 10 pg / ml was added on day 0 at 24 hour intervals.

25 d. Mean + mean error of 4-6 cultures.

e. nd = not done.

f. Percent inhibition DK 176020 B1 116

Antibody's ability to inhibit MLAM-1 shows that ICAM-1 monoclonal antibodies have therapeutic use in acute transplant rejection.

In addition, ICAM-1 monoclonal antibodies have therapeutic efficacy in immune-mediated diseases that depend on LFA-1 / ICAM-1 regulated cell-to-cell interactions.

The experiments described herein show that the addition of β monoclonal antibodies to ICAM-1 inhibits the mixed lymphocyte reaction (MUR) when added during the first 24 hours of the reaction. ICAM-1 is further upregulated on human peripheral blood monocytes during in vitro culture.

It has further been found that ICAM-1 is not expressed on quiescent human peripheral blood lymphocytes or monocytes. ICAM-1 is upregulated on the monocytes of cultured cells alone or cells that are co-cultured with unrelated donor cells in a mixed lymphocyte reaction using standard flow cytometric assays (Fig. 20). This up-regulation of ICAM-1 on monocytes can be used as an indicator of inflammation, especially if ICAM-1 is expressed on healthy monocytes from individuals with acute or chronic inflammation.

The specificity of ICAM-1 for activated monocytes and the ability of the antibody to ICAM-1 to inhibit an MLR suggests that ICAM-1 monoclonal antibodies must have a diagnostic and therapeutic option in acute transplant rejection and related immune-mediated diseases requiring cell-to-cell interactions. .

OK 176020 B1 117

Example 28

Synergistic effects of the combined administration of anti-ICAM-1 and anti-LFA-1 antibodies.

As shown in Example 27, MLR is inhibited by anti-5 ICAM-1 antibody. In addition, MLR can be inhibited by the anti-LPA-1 antibody. To determine whether the combined administration of anti-ICAM-1 and anti-LFA-1 antibodies would have an enhanced or synergistic effect, an MLR test (performed as described in Example 27) was performed in the presence of different concentrations. of the two antibodies.

This MLR test showed that the combination of anti-ICAM-1 plus' anti-LFA-1, at concentrations where no antibody alone dramatically inhibits MLR, is significantly 15 more potent in inhibiting the MLR reaction (Table 18).

This result indicates that therapies that involve the co-administration of anti-iCAM-i antibody (or fragments)

I I

! and anti-LFA-1 antibody (or fragments thereof) have the capacity to provide an improved anti-inflammatory therapy. Such improved therapy enables the administration of lower doses of antibody than would otherwise be therapeutically effective, and this is of importance where high concentrations of individual antibodies induce an anti-idiotypic reaction.

DK 176020 B1 118

Table 18 Effect of Different Doses of Anti-ICAM-1 and (R3.1) Anti-LFA-1 on Mixed Lymphocyte Reaction.

5% inclusion

Concentration (pg / ml)

Anti-ICAM-1 (R6-5-D6)

Anti-LFA-1 0 0.004 0.02 0.1 0.5 2.5 100.0 0 7 31 54 69 70 0.0008 1 7 28 48 62 71 0.004 0 13 30 50 64 72 0.02 29 38 64 75 84 86 0.1 92.5 90 91 92 92 92 150.5 93 90 90 92 93 91

Example 29

Effect of anti-ICAM-1 antibody on suppressing the rejection of transplanted allogeneic organs.

To demonstrate the effect of anti-ICAM-1 antibody in suppressing the rejection of an allogenically transplanted organ, Cynomolgus monkeys were transplanted with allogeneic kidneys in accordance with the procedure described by Cosimi et al. (Transplant. Proc. 13; 499-503 (1981)) with the change that valium and ketamine were used as anasthesla.

Thus, the kidney transplant was performed essentially as follows. Heterotropic renal allograft transplation was performed in 3-5 kg of Cynomolgus monkeys, essentially as described by Marquet (Marguet et al ♦,

Medical Primatology, Part II, Basel, Karger, p 125 (1972)) after induction of anesthesia with valium and 119.

DK 176020 B1 ketamine. End-to-side anastomoses of donor kidney vessels on a piece of aorta or vena cava were constructed relieving the use of the 7-0 Prolene suture. Donor ureter was spawned and implanted into the bladder at each extra-5 straval access (Taguchi, Y., et al., 1 Dausset et al. (Eds).) In: Advances In Transplantation, Baltimore, Williams & Wilkins, p. 393 (196Θ)). Renal function was evaluated by weekly or bi-weekly serum creatinine assays. In addition, frequent 10 allotransplant blocks were obtained for histopathological examinations, and complete autopsy was performed on all non-surviving recipients. In most recipients, bilateral nephrectomy was performed at the time of transplantation and subsequent uremic death was considered the endpoint of allotransplant survival. In some recipients, unilateral native nephrectomy and contralateral ureteral ligation were performed at the time of transplantation. When allotransplant rejection occurred, the ligature of the 20 autologous ureter was removed, resulting in restoration of normal renal function and the possibility of continuing immunologic registration in the receiving animal.

Monoclonal antibody R6-5-D6 was administered daily for 12 days, starting two days before transplantation, at a dose of 1-2 mg / kg / day. Serum concentrations of creatinine were periodically tested to detect rejection. The effect of anti-ICAM-1 antibody on the immuno-system rejection of the allogenic kidneys is shown in Table 15.

DK 176020 B1 120

Table 15

Survival time for allogeneic renal recipients.

Control- Survival- R6-5-D6 be- Survival- 5 .be ~ # _time (days) _treated monkey time (days) 5 1 8 1 20 2 11 2 8 3 11 3 30 10 4 10 4 31 5 9 5 11 6 10 6 23b 15 a The animals were given 1-2 mg / kg / day for 12 days starting 2 days before transplantation. b Still 1 live on April 8, 1988._

The results show that R6-5-D6 was effective at prolonging the life of monkeys receiving allogeneic renal transplants.

Claims (13)

121 DK 176020 B1 1. Monoclonal antibody, characterized in that it is R6-5-D6 capable of binding to a molecule selected from the group consisting of ICAM-5 and functional derivatives of ICAM-1. An antibody according to claim 1, characterized in that the molecule is capable of binding to a receptor found on the surface of a lymphocyte and that binding of the antibody to the molecule weakens the molecule's ability to bind to the receptor molecule of the lymphocyte. An antibody according to any one of claims 1-2, characterized in that it is in labeled form. A hybridoma cell, characterized in that it is capable of producing the monoclonal antibody according to any one of claims 1, 2 or 3. 5. A hybridoma cell, characterized in that it is capable of producing the monoclonal antibody R6-5-D6, the cell being ATCC HB 9580. A fragment of the antibody according to any one of claims 1-3, characterized in that it is capable of binding to the molecule. A fragment of the antibody according to claim 6, characterized in that it is in labeled form. A method for diagnosing the present room and locating an ICAM-1-expressing tumor cell in a mammal or human being suspected of containing such a cell, characterized in that it comprises: (a) incubating a tissue sample from the subject; with a mixture containing a detectably labeled binding ligand capable of binding to ICAM-1, the ligand being selected from the group consisting of an antibody according to any of claims 1-3 and a fragment of an antibody. according to any one of claims 6 or 7, wherein the fragment is capable of binding to ICAM-1 and 5 (b) detecting the binding ligand. Pharmaceutical composition, characterized in that it contains an antibody or toxin-derived antibody according to any one of claims 1-3, which is capable of binding to a molecule selected from An ICAM-1 and a functional derivative of ICAM-1, or a fragment of an antibody or toxin-derived fragment of an antibody according to any one of claims 6 or 7, wherein the fragment is capable of binding to a molecule selected from ICAM. -1 and a functional derivative of ICAM-1, together with an inert pharmaceutical carrier. Use of an antibody or toxin-derived antibody according to any one of claims 1-3 capable of binding to a molecule selected from An ICAM-1 and a functional derivative of ICAM-1, or a fragment of an antibody or toxin-derived fragment of an antibody according to any one of claims 6 or 7, which is capable of binding to a molecule selected from ICAM-1. and a functional derivative of ICAM-1, for the preparation of a pharmaceutical composition for treating inflammation arising from a response of the specific defense system of a mammal or human, or for suppressing the growth of an ICAM-1 expressing agent. tumor or an LFA-1-expressing tumor.