IE83840B1 - Intercellular adhesion molecules, and their binding ligands - Google Patents

Abstract

ABSTRACT The present invention relates to intercellular adhesion molecules (ICAM—1) which are involved in the process through which lymphocytes recognize and migrate to sites of inflammation as well as attach to cellular substrates during inflammation. The invention is directed toward such molecules, screening assays for identifying such molecules and antibodies capable of binding such molecules. The invention also includes uses for adhesion molecules and for the antibodies that are capable of binding them.

Description

TITLE OF THE INVENTION: INTERCELLULAR ADHESION MOLECULES: AND THEIR BINDING LIGANS BACKGNOUND 0F‘THE INVENTION Field of the Invention The present invention relates to intercellular adhesion molecules ICAM—l which are populations of lymphocytes recognize and adhere to cellular substrates such as involved in the process through which so that they may migrate to sites of inflammation and interact with cells during inflammatory reactions. The present invention additionally relates to ligand molecules capable of’ binding to such intercellular adhesion molecules, to a screening assay for these ligands, and to uses for the intercellular adhesion molecule, the ligand molecules, and the screening assay.

Description of the Related Art Leukocytes must be able to attach to cellular substrates in order to properly defend the host against foreign invaders such as bacteria or viruses. An excellent review of the defense system is provided by "attachments were - g _ Eisen, H.N., (1g;__jfigrgbjQ1ggy, 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 circulation to sites of ongoing inflammation. Furthermore, they must attach to antigen~ presenting cells so that a normal specific immune respdhse can occur, and finally, they must attach to appropriate target!cells so that lysis of virally-infected or tumor cells can occur._ identified which bound to leukocyte surfaces and inhibited the attach— Briefly, et al., Fed. Proc.

(Springer, T.A., et al. Immunol. Rev- to I80 kd (Springer, T., Fed. Proc. gg:2660—2663 (L985)). Although the in cellular adhesion _ 3 _ alpha subunits of the nmmbrane proteins do not share the extensive homology shared by the beta subunits, close analysis of the alpha subunits of the glycoproteins has revealed that there are substantial similarities between them. Reviews of the similarities.between the alpha and beta subunits of the LEA—l related glycoprpteins are provided by Sanchez—Hadrid, F. et al., (J. Exper. Med. l58:S86—602 (1983); Q4: Furthermore, these individuals were unable normal counterparts whose family of molecules had been antagonized by antibodies. to mount a normal immune response due to an inability of their cells to U.C., et al., Fed. Proc. (1985)). These data show that immune reactions are mitigated when lymphocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules of the LFA-1 family“ The expression of a recently identified ligand of LFA—l, the (Anderson, intercellular adhesion molecule—1 (ICAM-1) was investigated in various cell types in response to cytokines (Rothlien R. : 1665-1669 (1988)). The induction of et al, J. Immunol, lCAM—1 was neutralised by cytokine specific antisera and some pharmacological agents. Cyclohexamide increased the expression of ICAM—1 on chondrosarcoma cells but had little or no effect on carcinoma cells. This data indicated ‘ different mechanisms in the regulation and expression of ICAM—1 on the Various cell types.

Thus, in summary, the ability of lymphocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells)5 This adherence has been found to require cell~cell contacts which involve specific receptor molecules present on the cell surface of the lymphocytes. a-lymphocyte to adhere to other lymphocytes or to endothelial, and The cell surface receptor molecules have These receptors enable other non-vascular cells. been found to be highly related to one another. lymphocytes lack these cell surface receptor molecules exhibit chronic and recurring infections, as well as other clinical symptoms including Humans whose defective antibody responses. T Since lymphocyte adhesion is involved in the process through which Foreign tissue is identified and rejected, an understanding of this process is of significant value in the fields of organ transplantation, tissue grafting, allergy and oncology. sumwzv or THE INVENTION The present invention relates to Intenzellularjldhesion H0lecule—1 (ICAH—l) as well as to its functional derivatiies ; to antibodies and fragiaénts of antibodies capable do; inhibiting the function «of ICAH-‘1_, and to ‘other inhibitors of ICAM—l function Theinventionincludestherapeutic uses for allof the 350V?‘ described molecules.

In detail, the invention is directed toward a method for treating inflammation resulting from a response of the specific defence system in a mammalian subject which comprises providing to a subject in need of such treatment an amount of an anti-inflammatory agent sufficient to suppress the inflammation; where in the anti—inflammatory agent is selected from the group consisting of: an antibody capable of binding to ICAM~1; a fragment of an antibody, the fragment being capable of binding to ICAM—1; ICAM—1; a functional derivative of ICAM-1; and of ICAM-1; and a non- immunoglobulin antagonist of ICAM-1.

The investigation further includes the above described method of treating inflammation wherein the non- immunoglobulin antagonist of ICAM—1 is a non-immunoglobulin antagonist of ICAM—1 other than LFA—1.

The invention includes a phamaceutical composition comprising: (a) an anti-inflammatory agent selected from the group Consisting of: an antibody capable of binding to ICAH-1; a firagment of an - antibody, the fragment being capable of binding to [CAM-1; {CAM-1; a functional derivative of ICAM-1; and a non-immunoglobulin antagonist of ICAM—l, and (b) at least one immunosuppressive agent selected from the group consisting of: dexamethesone, azatfiioprine and cyclosporin A- _ 5 - BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows in diagrammatic form the adhesion between a normal and an LFA~1 deficient cell.

Figure 2 shows in diagrammatic form the process of: normal/normal cell adhesion. I * Figure 3 shows the kinetics of cellular aggregation in the absence (X) or presence of 50 ng/ml of PMA (0). ‘ Figure 4 shows coaggregation ‘between [FA-I“ and LFA-1* cells.

Carboxyfluorescein diacetate labeled EBV-transformed cells (104) as designated in the figure were mixed with 105 unlabeled autologous cells After 1.5 h the labeled cells, in aggregates or free, were enumerated using the (solid bars) or JY cells (open bars) in the presence of PMA. qualitative assay of Example 2. The percentage of labeled cells in aggregates is shown. One representative experiment of two is shown.

Figure 5 shows the immunoprecipitation of ICAM—l and LFA—l from JY cells. Triton X-100 lysates of JV cells (lanes 1 and 2) or control lysis buffer (lanes 3 and 4) were immunoprecipitated with antibody capable of binding to ICAM—l (lanes 1 and 3) or antibodies capable of binding to LFA-1 (lanes 2 and 4). conditions; Panel 8 conditions. Molecular weight standards were run in lane S.

Panel A shows results under reducing shows results obtained under non—reducing Figure 6 shows the kinetics of IL-l‘and gamma interferon effects on ICAM-1 expression ‘on fibroblasts. dermal fibroblasts were grown to a density of 8 x 104 cells/0.32 cmz well.

IL-1 (10 U/ml, closed circles) or recombinant gama interferon (10 U/ml, open squares) was added, and at the indicated time, the assay was indirect binding assay was performed. The human dermal Human cooled to 4'C and an standard deviation did not exceed 10%.

Figure 7 shows the concentration dependence of IL-1 and gama Human dermal fibroblasts were grown to a circle), interferon effects on ICAM-1. density of 8 x 104 cells/0.32 cmz/well.‘ IL-2 (open square), recombinant mouse IL-1 (solid (open recombinant human IL-1 square), recombinant human gamma interferon (solid circles), and recombinant beta interferon (open triangle) were added at the indicated dilution and were incubated for 4 hours (IL-1) or 16 hours (beta and results are ‘the means from gama interferon); The indicated quadruplicate determinations; standard deviation did notiexceed 10%.

Figure 8 shows the nucleotide and amino acid 'sequence of ICAM—l CDNA. The first ATG is at positiong S8. Translated sequences corresponding to ICAM~1 tryptic peptides‘ are underlined. The "hydrophobic putative signaT peptide and transmembrane sequences have a bold underline. N-linked glycesylation sites are boxed. The polyadenylation signal AATAAA at position 2976 is over-lined. The sequence shown is for the HL-60 CDNA clone. The endothelial cell CDNA most of its length and showed only minor was sequenced over differences.

Figure 9 shows the ICAM—I homologous domains and relationship to the immunoglobulin supergene family. (A) Alignment of 5 homologous domains (01-5).

Residues conserved 2 or more times in NCAM domains, as well as resides conserved in domains of the sets C2 and C1 were aligned with the [CAM-1 The location of the predicted B strands in the [CAM- aoove the Two or more identical residues which aligned are boxed. internal repeats. 1 domain is marked with bars and alignments, and the known location of fl-strands in immunoglobulin C domains is marked with bars and capital letters below the alignment.

The position of the putative disulfide bridge within ICAM-1 domains is indicated by S-S. (B-D) Alignment of protein domains homologous to ICAM-1 domains; proteins were initially aligned by searching NBRF databases using the FASTP program. The protein sequences are MAG, lower case letters NCAM, T cell receptor a subunit V domain, IgMp chain and aB- glycoprotein.

Figure 10 shows a diagrammatic comparison of the secondary structures of ICAM-1 and MAG.

Figure 11 shows LFA—1—positive EBV—transformed B—lymphoblastoid cells binding to ICAM—l in planar membranes.

Figure 12 shows LFApositive T lymphoblasts and T lymphoma cells bind to ICAM-l in plastic~bound vesicles.

Figure 13 shows the inhibition of binding of JY B-lymphoblastoid cell binding to ICAM-1 in plastic-bound vesicles by pretreatment of cells or vesicles with monoclonal antibodies. s Figure 14 shows the effect of temperature lymphoblasts to [CAM-1 in plastic-bound vesicles.

Figure 15 shows divalent cation requirements for binding of T- lymphoblasts to ICAM—l in plastic-bound vesicles.

Figure 16 shows the effect of» anti—adhesion antibodies on the on binding of T- ability of peripheral blood mononuclear cells to proliferate in response to the recognition of the T—cell associated antigen OKT3.

"OKT3" indicates the addition of antigen.

Figure 17 shows the effect of anti—adhesion antibodies on the ability of peripheral blood mononuclear cells to proliferate in recognition of the non—specific T-cell mitogen, "CONA“ indicates the addition of concanavalin A. response to the concanavalin A.

Figure 18 shows the effect of anti-adhesion antibodies on the blood mononuclear cells to proliferate in ability of peripheral response to the recognition of the keyhole limpet hemocyanin antigen.

The addition of keyhole limpet hemocyanin to the cells is indicated by "KLH." Figure 19 shows the effect of ahti-adhesion antibodies on the ability of peripheral blood mononuclear cells 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." Figure 20 shows the binding of monoclonal antibodies RR1/1, R6.5, LB2, and CL203 to ICAM-1 deletion mutants- Figure 21 shows the binding of ICAM—1 deletion mutants to LFA—1.

Figure 22 shows the epitopes recognized by anti-ICAM-1 monoclonal antibodies RRI/1, R6.5, LB2, and CL203.

Figure 23 shows binding capacity of ICAM—l-domain 2 mutants to LFA- Figure 24 shows binding capacity of ICAM-1 domain 3 mutants to LFA- Figure 25 shows binding capacity of ICAM-1 domain 1 mutants to LFA- .

’ Figure 26 shows the alignment of ICAM amino-terminal domains.

DESCRIPTION OF THE PREFERRED EMBODIRENTS One aspect of the present invention relates to the discovery of a natural binding ligand to LFA-l. v Molecules such as those of LFA~l family, which are involved in the process of cellular adhesion are referred to as "adhesion molecules." The natural binding ligand of the present invention is designated ICAM—l is a 76-97 Kd The present invention is "Intercellular Aadhesion Molecule-1" or “ICAM—l." glycoprotein. ICAM-1 is not a heterodimer. directed toward ICAM-1 and its "functional derivatives.“ ICAM—1 is activity (either functional or structural) activity of ICAM-1. "fragments," A "functional a compound which posesses a biological that is The term “functional derivative“ of substantially similar to a biological intended to "variants," derivatives“ is include the "analogs," or “chemical derivatives“ of a molecule. molecule such as ICAM~1, is meant to refer to any polypeptide subset of the molecule. Fragments of ICAM-1 whidh have ICAM—1 activity and which are soluble (i.e not membrane bound) are especially preferred. A "variant" of a molecule such as ICAM-1 is meant to refer to a molecule substantially similar in structure and function to either the entire molecule, or to a fragment thereof. is said to be "substantially similar" to another molecule if both ‘molecules have substantially similar structures or if both molecules possess a similar biological activity. Thus, provided that two molecules possess a they are considered variants as that term is used A “fragment” of a A molecule similar activity, herein even if the structure of one of the molecules not found in the other, or if the sequence of amino acid residues is not identical. An "analog" of a molecule such as ICAM-1 is meant to refer to a molecule substantially similar in function to either 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 not normally a part .of the molecule. Such"moieties may improve the nmlecule’s solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, Moieties’ capable of mediating such effects are disclosed in Remington's ‘Pharmaceutical Sciences (1980). derivatized“ molecules constitute a special class of “chemical deriva- etc.

"Toxin— tives." A 'toxin—derivatized” molecule is a molecule (such as ICAM~l or an antibody) which contains a toxin moiety. molecule to a cell brings the toxin moiety into close proximity with The binding of such a the cell and thereby promotes cell death. Any suitable toxin moiety it is preferable to employ toxins such as, the diphtheria toxin, may be employed; however, for example, the ricin toxin, radioisotopic toxins, membrane—channel-forming toxins, etc. Procedures 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 are naturally expressed on the surfaces of lymphocytes.

Thus, the introduction of such cells into an appropriate animal, as by intraperitoneal etc., will result in the production of antibodies capable of binding to ICAM—l or members of the LFA-1 family of molecules. If desired, the serum of such an animal may be removed and used as a source of polyclonal antibodies capable of binding these molecules. It is, however, preferable to remove splenocytes from such animals, to fuse such spleen cells with a myeloma cell line and to permit such fusion cells to form a hybridoma cell which secretes monoclonal antibodies capable of binding ICAM-l or members of the LFAs1 injection, family of molecules.

The hybridoma cells, obtained in the manner described above may be screened by a variety of methods to identify desired hybridoma cells that secrete antibody capable of binding either to ICAM—1 or to members of the LFA-1 family of molecules. molecules are identified by their ability to inhibit the aggregation of Epstein-Barr virus-transformed cells. Antibodies capable of inhibiting such aggregation are then further screened to determine whether they inhibit such aggregation by binding to ICAM-1, or to a member of the LFA-1 family of molecules. Any means capable of distinguishing ICAM-I from the LFA—1 family of molecules may be employed in such a screen.

Thus, for example, the antigen bound by the antibody may be analyzed as by immunoprecipitation and polyacrylamide gel electrophoresis. If the bound antigen is a member of the LFA-1 family of molecules then the In a preferred screening assay, such immunoprecipitated antigen will be found to be a dimer, whereas if the bound antigen is ICAM-1 a single molecular weight species will have been immunoprecipitated. Alternatively, it is possible to distinguish between those antibodies which bind to members of the LFA-1 family of molecules from those which bind [CAM-1 by screening for the ability of the antibody 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 that the antibody is capable of binding LFA—1. The absence of such binding is indicative of an antibody capable of recognizing ICAM-1. The ability of an antibody to bind to a cell such as a granulocyte may be detected by means commonly employed by those bf ordinary skill. Such means include immunoassays, cellular agglutination, filter binding studies, indicates antibody precipitation, etc. invention of the present may The anti-aggregation antibodies alternatively be identified by measuring their ability to differen- tially bind to cells which express ICAM—1 (such as activated endothelial cells), and their inability to bind to cells which fail to express [CAM-1. As will be readily appreciated by those of ordinary skill, the above assays may be modified, or performed in a different sequential order to provide a variety of potential screening assays, each off which is capable of identifying and discriminating between antibodies capable of binding to ICAM—l versus members of the LFA-1 family of molecules.

The anti-inflammatory agents of the present invention may be obtained by natural processes (such as, for example, by inducing an animal, plant, fungi, bacteria, etc., to produce a non5lmmunoglobulin antagonist of ICAM-1, or by inducing an animal to produce polyclonal antibodies capable of binding to ICAH-1); by synthetic methods (such as, for example, by using the .Merritield, method for synthesizing polypeptides to synthesize jCAMél, functional derivatives of ICAM-I, or protein antagonists of ICAM-1 ' (either immunoglobulin)); by hybridoma technology (such as, for example, to or by immunoglobulin or non- produce monoclonal antibodies capable of binding to ICAMVI); recombinant technology (such as, for example, to produce the anti- inflammatory agents of the present invention in diverse hosts (i.e., etc.), or from cultured mamalian cells, The choice of which method to yeast, bacteria, fungi, recombinant plasmids or viral vectors). employ will depend upon factors such as convenience, desired yield, etc. It is not necessary to employ only one of the above—described methods, or technologies to produce a particular anti- processes, inflammatory agent; the technologies may be combined in order to obtain a particular anti- above-described processes, methods, and inflammatory agent.

A. Identification of the LFA-1 Binding Partner (ICAM-1) 1. Assays of LFADependent Aggregation herein by reference). Thus, the extent of LFA-1—dependent binding may _ 13_- be determined by assessing the extent of spontaneous or phorbol ester- dependent aggregate formation. molecule.

The cells should be cultured under conditions culture medium Laboratories, NY). suitable for mammalian cell proliferation (i.e., at a temperature of generally 37'C, in an atmosphere of 5% C02, at a relative humidity of 95%, etc.).

. LFA-1 Binds to ICAM-1 _.14 - aggregates failed to form if these cells were incubated in the presence of anti-LFA-1 antibodies. Thus, although the aggregation required LFA- 1, the ability of LFA-I-deficient cells to form aggregates with LFA containing cells indicated that the [FA—l binding partnergwas not LFA-1 but was rather a previously undiscovered cellular adhesion molecule.

Figure 1 shows the mechanism of cellular adhesion.

. Intercellular Adhesion Molecule=l (ICAM-1)‘ monoclonal antibodies were prepared from spleen cells of mice immunized is herein incorporated by reference. molecule, with cells from individuals genetically deficient in LFA-l expression.

Resultant antibodies were screened for their ability to inhibit the aggregation of LFA-l-expressing cells (Figure 2). In detail, the ICAM- 1 molecule, mice were immunized with EBV-transformed B cells from LAD patients which do not express the LFA-l antigen. The spleen cells from these animals were subsequently removed, fused with myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. E8V— transformed B cells from normal individuals which express LFA-1 were then antibody of the hybridoma cell in order to identify any monoclonal antibody which was capable of inhibiting the phorbol ester mediated, LFA-1 dependent, spontaneous aggregation of the EBV-transformed B cells. Since the hybridoma cells were derived from cells which had never encountered the LFA-1 antigen no monoclonal antibody to LFA-1 was produced_ Hence, any antibody found to inhibit aggregation must be capable of binding to an antigen that, although not LFA—l, participated in the LFA-1 adhesion process. Although any method of obtaining such monoclonal antibodies may be employed, it is preferable to obtain lCAM-l—binding monoclonal antibodies by immunizing BALB/C mice using the routes and schedules incubated in the presence of the monoclonal (In a preferred method for the.generation and detection if antibodies capable of binding to ICAM-1, mice are immunized. with either EBV- transformed B cells which (express both ICAM-1 and LFA-1 or more preferably with TNF-activated endothelial cells which express ICAM-1 but not LFA-1. In a most:preferred method for generating hybridoma cells which produce anti—ICAM-1 antibodies, a Balb/C mouse was sequen- tially imunized with JY cells and with differentiated U937 cells (ATCC CRL-1593). cells The spleen cells from such animals are removed, fused with myeloma and permitted to develop into antibodyvproducing .tonsils, lymph nodes, and Peyer’s patches. _ 16 - alternative is to immunoprecipitate from cells expressing [CAM-1, LFA~ 1, or both, that inhibit the LFA—1 aggregation of cells, such as JY cells, and through SDS-PAGE or an using antibodies dependent ‘equivalent ‘method determine "some molecular: charactergstic of the" molecule precipitated with the antibody. If the characteristic is the same as that of ICAH-1 then the antibody can be assumed to be an anti- ICAM—l antibody. ~ ‘ Using monoclonal antibodies prepared in the manner described above, the ICAM-I cell surface molecule :was purified, and characterized.

ICAM—1 was purified from human cells or tissue using monoclonal antibody affinity chromatography. In such a method, a nmnoclonal antibody reactive with ICAMv1 is coupled to an inert column matrix. the ICAM—l molecules present are adsorbed and retained by the matrix. when a cellular lysate is passed through the matrix By altering the pH or the ion concentration of the column, the bound ICAM-1 molecules may be eluted from the column- Although any suitable matrix can be employed, it is preferable to employ sepharose (Pharmacia) as the matrix material- The formation of column matrices, and their use in protein purification are well known in the art.

In a manner understood by those of ordinary skill, the above- described may be used to rdentify compounds capable of attenuating or inhibiting the rate or extent of cellular adhesion.

ICAM-1 is a hematopoietic cells such as endothelial epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal centered B cells and dendritic cells in ICAM-1 is highly expressed in lymph nodes and assays cell surface glycoprotein expressed on non- vascular cells, thymic in T cell areas ICAM-1 Phorbol on vascular endothelial cells tonsils showing reactive hyperplasia. is expressed in low amounts on peripheral blood lymphocytes. ester-stimulated differentiation of some myelomonocytic cell lines greatly increases ICAM-1 expression. Thus, ICAM-1 is preferentially expressed at sites of inflammation, and is not generally expressed by quiescent cells.

ICAM-ll expression on dermal "fibroblasts is increased threefold to‘ fivefold by either interleukin_1 or gamma interferon at levels of 10 U/ml over a period of 4 or 10 hours, respectively. The induction is dependent on protein and mRNA synthesis and is reversible.

ICAM—l displays molecular weight heterogeneity in different cell types with a nmlecular weight of 97 kd on fibroblasts, 114 kd on the myelomonocytic cell line U937, and'90 kd on the B lymphoblastoid cell JY. ICAM-1 biosynthesis has been found to involve an approximately 73 kd intracellular precursor. The non—N-glycosylated form resulting from tunicamycin treatment (which inhibits glycosylation) has a nmlecular weight of 55 kd.

ICAM-1 isolated from phorbol ester stimulated U937 cells or from fibroblast cells yields an identical major product having a molecular weight of 60 kd after chemical deglycosylation. ICAM-1 monoclonal antibodies interfere with the adhesion of phytohemagglutinin blasts to LFA-1 deficient cell Pretreatment of fibroblasts, but not with monoclonal ICAM—1 Pretreatment of lymphocytes, lines. lymphocytes, antibodies capable of binding inhibits lymphocyte-fibroblast adhesion. but not fibroblasts, with antibodies against LFA—l has also been found to inhibit lymphocyte-fibroblast adhesion. hematopoietic cells such as tissue macophages,P mitogen—stimulated T DOD‘ cells cells, thymic cells, other epithelial Vvbenign inflammatory lesions such as allergic eczema, lymphocyte blasts, and germinal center B-cells and dendritic cells in tonsils, lymph nodes and Peyer’s patches (Dustin, M.L., et. al., Q; ICAM—1 is present on Allergic skin reactions keratinocytes. reveal ICAM-1 expression on the keratinocytes. keratinocytes from biopsies of skin lesions from various dermatological disorders and ICAM-1 expression is induced on lesions from allergic patch tests while keratinocytes from toxic patch test lesions failed to express iCAM—l.

ICAM—l is, therefore, a cellular substrate to which lymphocytes can attach, so that the lymphocytes may migrate to sites of inflammation and/or carry out various this inflammation. Such functions include the production of antibody, lysis effector functions contributing to of virally infected target cells, etc. The term "inflammation," as used herein, is meant to include reactions of the specific and non— specific defense systems. As used herein, the term “specific defense system" is intended to refer to that component of the imune system that reacts to the presence of specific antigens. to result from a response of the specific defense system if the inflammation is caused by, mediated by, or associated with a reaction Inflammation is said of the specific defense system. Examples of inflammation resulting from a response of the specific defense system include the response to antigens such as rubella virus, autoimmune diseases, delayed type hypersensitivity response mediated by T-cells (as seen, for example in individuals who test ‘positive’ in the Mantaux test), psoriasis, etc.

Q - 19 _ -.

-—-. A “non—specific defense system reaction" is a response‘mediated by leukocytes incapable of immunological memory. Such cells include granulocytes and macrophages. As used herein, inflammation is said to result from a response of the non-specific defense system, if the inflammation is caused by, mediated by, or associated with a reaction of the non—specific defense system. Examples of inflammation which result, at least in part, from a reaction of the non—specific defense system include inflammation associated with conditions sdch as: asthma; adult respiratory distress syndrome (ARDS) or multiple organ injury syndromes secondary to septicemia or trauma; reperfusion injury 'of myocardial or other tissues; acute glomorulonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other central nervous system inflammatory hemodialysis; leukapheresis; ulcerative disorders; thermal injury; colitis; Crohn's disease; necrotizing enterocolitis; granulocyte transfusion associated syndromes; and cytokine-induced toxicity.

In accordance with the present invention, lCAM—l functional derivatives, and especially such derivatives which comprise fragments or mutant variants of ICAM—1 which possess domains 1, 2 and 3 can be used in the treatment or therapy of such reactions of the non—specific defense system. More preferred for such treatment or therapy are {CAM-1 fragments or mutant variants which contain_domain 2 of ICAM—1.

Most preferred for such treatment or therapy are ICAM-1 fragments or mutant variants which contain domain 1 of ICAM-1.

C. Cloninq of the IChM—l Gene Any of a variety of procedures may be used to clone the ICAM-1 gene.

One such method entails analyzing a shuttle vector library of CUNA inserts (derived from an ICAM—l expressing cell) for the presence of an insert which contains the ICAM-1 gene. Such an analysis may be conducted by transfecting cells with the vector and then assaying for ICAM—1 expression. The preferred method for cloning this gene entails determining the amino acid sequence of the ICAM-1 molecule. To accomplish this task [CAM-1 protein may be purified and analyzed by automated sequenators. Alternatively, the molecule may be fragmented as with cyanogen bromide, or with proteases such as papain, (1982); Liu, C. et 31., Int. J. Peot.lProteinARes. g;:2o9-215 (1983‘)).' Although it is possible to determine the entire amino acfid sequence of ICAM—1, it is preferable to determine the sequence of peptide fragments of the molecule. If the peptides are greater than 10 amino acids long, the sequence information is generally sufficient to permit one to clone a gene such as the gene for:ICAM-1.‘ The sequence of amino acid residues in a peptide is designated herein either through the use of their comonly employed 3-letter designations or by their single~letter designations. A listing of these 3—letter and 1-letter designations may be found in textbooks such as Biochemistry, Lehninger, A., Worth Publishers, New York, NY (1970). when such a sequence is listed vertically, the amino terminal residue is intended to be at the top of the list, and the carboxy terminal residue of the peptide is intended to be at the bottom of the list.

Similarly, when listed horizontally, the amino terminus is intended to be on the left end whereas the carboxy terminus is intended to be at the right end. The residues of amino acids separated by hyphens. Such hyphens are intended solely to facilitate the presentation of a sequence. in a peptide may be As a purely illustrative example, the amino acid sequence designated: ‘ -Gly-Ala-Ser—Phe— indicates that an Ala residue is linked to the carboxy group of Sly, and that a Ser residue is linked to the carboxy group of‘ the Ala residue and to the amino group of a Phe residue. The designation further indicates that the amino acid sequence contains the tetrapeptide Gly~Ala-Ser-Phe. The designation is not intended to limit the amino acid sequence to this one tetrapeptide, but is intended to include (1) the tetrapeptide having one or more amino acid residues linked to either its amino or carboxy end, (2) the tetrapeptide having one or more amino acid residues linked to both its amino and its carboxy ends, (3) the tetrapeptide having no additional amino acid residues. ‘ Once one or more suitable peptide fragments have been sequenced, the DNA sequences capable "of encoding them are examined. genetic code is degenerate, more.than one codon may be us:d to encode a particular amino acid (Watson, J.D., In: Molecudar Biology of the Qggg, 3nd Ed., H.A. Benjamin, Inc., Menlo ?ark, CA (1977), pp. 356- 357). The peptide fragments are analyzed -to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon. Although occasionally such amino acid sequences may be encoded by only a single oligonucleotide, frequently the amino acid sequence oligonucleotides. encoded by any of a set of similar members of the set can be importantly, whereas all of the contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same nucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains a nucleotide sequence that is identical to the nucleotide sequence of this gene.. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in‘which one would employ a single oligonucleotide to clone the gene that encodes the peptide- In a manner exactly analogous to that described above, one may employ an oligonucleotide (or set of oligonucleotides) which have a nucleotide sequence that is complementary to the oligonucleotide sequence or set of sequences that is capable of encoding the peptide fragment.

A suitable oligonucleotide, capable of encoding a fragment of the ICAM—1 gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified (using the above—described procedure), synthesized, and or set of oligonucieotides which is Because" the ' 35 hybridized, by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from human cells which are capable of expressing ICAM-1 gene sequences. acid hybridization are disclosed by Haniatis, T. et al., In: Cloning, a Laboratory Manual, icoldspring Harbor, NY (l982), and by Haymes, B.D. et al., In: Nucleic Acid Hvbrization. a Practical Approach, IRL Press, Washington, DC (1985), which references are herein incorporated by reference. A The. source~ of: DNA or cDNA used will preferably have been enriched for {CAN-l sequences. Such enrichment can most easily be obtained from cDNA obtained by extracting RNA from cells cultured under conditions which induce ICAM—1 synthesis (such as Techniques of nucleic Molecular U937 grown in the presence of phorbol esters, etc.). such as, or similar to, those described above have In a preferred alternative way of 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 which binds to anti-ICAM-1 antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as ICAM-1 or fragments of ICAM—l. lechniques cloning of genes for human aldehyde et al., Eur. Mol. Biol. Orqan. J.

;, Proc. eukaryotic cells to produce supra, and are well known in the art.

. Uses of Assays of LFA—l Dependent Aqqreqation The above-described assay, capable of measuring LFA-1 dependent aggregation, may be employed. to identify agents Which act as antagonists to inhibit the extent of LFA-1 dependent aggregation. Such antagonists may act by impairing the ability of LFA-1 or of ICAM-1 to Thus, such agents include immunoglobulins such as either LFA-1 or ICAM—l. chemical) may be mediate aggregation. an antibody capable Additionally, non—imunoglobulin ‘(i.e., examined, using the above~described assay, to determine whether they of. binding to agents are antagonists of LFA-1 aggregation.

E. Uses of Antibodies Capable of Binding to ICAM—l Receptor Proteins . Anti-Inflammatory Agents (1986)), and effector functions of all leukocytes such as lytic activity of cytotoxic T cells (Krensky, A.M., et al., J . Immunol.

As discussed above, the binding of ICAM-1 molecules to the members of LFA-1 family of molecules is of central importance in cellular adhe- sion. Through the process of adhesion, lymphocytes are capable o continually ‘monitoring anlanimal for the presence" of foreign antigens." lllthough these processeslare normally desirable, they are also the cause of organ transplant rejection, tissue graft rejection and many autoimmune diseases. Hence, any means capable of attenuating or inhibiting cellular adhesion would be highly desirable in recipients of organ transplants, tissue grafts or‘ autoinmune patients.

Monoclonal antibodies capable ‘of binding to ICAM~l are highly suitable as anti-inflammatory agents in a mammalian subject.

Significantly, such agents differ from general anti-inflammatory agents in that they are capable of selectively inhibiting adhesion, and do not offer other side effects such as nephrotoxicity which are found with conventional agents. Monoclonal antibodies capable of binding to ICAM- 1 can therefore be used to prevent organ or tissue rejection, or modify autoimmune responses without the fear of such side effects, in the rmammalian subject.

Importantly, the use of monoclonal antibodies capable of recognizing ICAM—1 may permit one to perform organ transplants even between individuals having HLA mismatch.

. Suppressors of Delayed Type Hypersensitivity Reaction Since [CAM-I molecules are expressed mostly at sites of inflammation, such as those sites involved in delayed type hypersensitivity reaction, antibodies (especially monoclonal antibodies) capable of binding to ICAM-l molecules have therapeutic potential in the attenuation or elimination of such reactions. This potential therapeutic use may be exploited in either of two manners.

First, a composition containing a monoclonal antibody against ICAM-1 may be administered to a patient experiencing delayed type hyper- sensitivity reaction. For example, such compositions might be provided ‘conjunction with’ an antigen in order‘ to "prieventia _ 25 _ to a individual who had been in contact with antigens such as poison ivy, poison oak, etc. In the second embodiment, the monoclonal antibody capable of binding to ICAM—l is administered to a patient in inflammatory reaction. Thus, .the additional administ:ation of an antigen with an ICAMbinding monoclonal antibody may temporarily tolerize an individual to subsequent presentation of that antigen.

. Therapy for Chronic Jnflammatory Disease Since LAD patients that lack LFA-1 do not mount an inflammatory response, it is believed that antagonism of LFA—1’s natural ligand, The ability of antibodies against [CAM-1 to inhibit inflammation provides the basis ICAM—l, will also inhibit an inflammatory response. for their therapeutic use in the treatment of‘ chronic inflammatory diseases and autoimune diseases such as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), forms of diabetes Such antibodies may also be employed as a multiple sclerosis, some Reynaud’s syndrome, rheumatoid arthritis, etc. therapy in the treatment of psoriasis. In general, the monoclonal antibodies capable of binding to ICAM—l may be employed in the treatment of those diseases currently treatable through steroid therapy.

. Diagnostic and Prognostic Applications Since ICAM-l is expressed mostly at sites of inflammation, monoclonal antibodies capable of binding to ICAM«l may be employed as a means of imaging or visualizing the sites of infection and inflammation in a patient. In such a use, the monoclonal antibodies are detectably labeled, through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc.

Procedures for accomplishing such labeling are well known to the art. subsequent‘ ‘Science ;gg:29S'-"297 (1930)). _ 26 _ ' The presence of inflammation may also be detected through the use of binding ligands, such as mRNA, CDNA, or DNA which bind to ICAM-1 gene sequences, or to ICAM-l mRNA sequences, of cells which express ICAM-1.

'Techniques for performing such hybridization assays are described by Maniatais, T. (supra). , ‘ The detection of foci of such; detectaoly labeled antibodies is indicative of a site of inflammation or tumor development- In one this samples of tissue or blood and incubating such samples in the presence embodiment, examination for inflammation is done by removing of the detectably labeled antibodies. In a preferred embodiment, this technique is done in a non—invasive manner through the use of magnetic imaging, fluorography, etc. Such a diagnostic test may be employed in monitoring organ transplant recipients for early signs of potential tissue rejection- Such assays may also be conducted in efforts to determine an individual’s predilection to rheumatoid arthritis or other chronic inflammatory diseases.

. Adjunct to the Introduction of Antigenic Material Administered for Therapeutic or Diagnostic Phrposes Immune responses to therapeutic or diagnostic agents such as, for example, bovine insulin, interferon, tissue-type plasminogen activator or murine monoclonal antibodies substantially impair the therapeutic or diagnostic value of such agents. and can, in fact, causes diseases such as serum sickness. Such a situation can be remedied through the use of the antibodies of the present invention. In this embodiment, such antibodies would be administered in combination with the therapeutic or The addition of the antibodies prevents the and therefore prevents the diagnostic agent. recipient from recognizing the agent. recipient from initiating an immune response against it. The absence of such an immune response results in the ability of the patient to receive additional administrations of the therapeutic or diagnostic agent.

F. Uses of Intercellular Adhesion Molecule-1 (ICAM-ll [CAM-l is a binding partner of LFA-ell. _‘As such, ICAM-1 or its functional derivatives may be employed interchangeably with antibodies capable of binding to LFA-1 in the treatment of disease. Thus, in solubilized form, such molecules may be employed to inhibit inflamma- tion, organ rejection, graft rejection, etc. ICAM-1, or its functional derivatives may be used in the same manner as anti-[CAM antibodies to decrease the imunogenicity of therapeutic or diagnostic agents. lCAM—1, its functional derivatives, and its antagonists may be used to block the metastasis or proliferation of tumor cells which express either ICAM-1 or LFA-1 on their surfaces. A variety of methods may be used to accomplish such a goal. For example, the migration of hematopoietic cells requires LFA-1~ICAM—l binding. binding therefore suppress this migration and block the metastasis of toxin-derivatized Antagonists of such tumor cells of leukocyte lineage. Alternatively, molecules, capable of binding either ICAM-I or a member of the LFA-1 family of molecules, may be administered to a patient. when such toxin~derivatized molecules bind to tumor cells that express ICAM-l or a member of the LFA—l family of molecules, the presence of the toxin kills the tumor cell thereby inhibiting the proliferation of the tumor.

G. Uses of Non-Immunoqlobulin Antagonists of [CAM-1 Dependent Adhesion ICAMdependent adhesion can be inhibited by non-imunoglobulin antagonists which are capable of binding to either ICAM-1 or to LFA-1.

One example of a non-immunoglobulin antagonist of ICAM-1 is LFA-1. An example of a non~immunoglobulin antagonist which binds to LFA—1 is ICAH-1. Through the use of the above-described assays, additional non- immunoglobulin antagonists can be identified imunoglobulin antagonists of ICAM-I dependent adhesion may be used for and purified. Non- ’the same purpose as antibodies to LFA—l or antibodies to ICAH—1.

H. Administration of the Compositions of the Present Invention The therapeutic effects of ICAM-1 may be obtained by providing to a patient the entire ICAM-1, molecule, or any therapeutically active peptide fragments thereof. » ICAM—1 and its synthetically, through the use of recombinant DNA technology, or by functional derivatives may be obtained either proteolysis. The therapeutic advantages of ICAMAI may be augmented through the use of functional derivatives of ICAM~l possessing addi- tional amino acid residues added to enhance coupling to carrier or to enhance the activity of the ICAM-1. is further intended to include functional derivatives of ICAM—l which The scope of the present invention lack certain amino acid residues, or which contain altered amino acid residues, so long as such derivatives exhibit the capacity to affect cellular adhesion.

Both the antibodies of the present invention and the ICAM—l molecule disclosed herein are said to be free of natural contaminants“ if preparations which contain them are substantially free of materials with which these products are normally and naturally 'substantiallv found.

The present invention extends to antibodies, and biologically active fragments thereof, (whether polyclonal or monoclonal) which are capable of binding to ICAM-1. Such antibodies may be produced either by an animal, or by tissue culture, or recombinant DNA means.

In providing a patient with antibodies, or fragments thereof, capable of binding to [CAM-1, or when providing ICAM—l (or a fragment, variant, or derivative thereof) to a recipient patient, the dosage of vary depending upon such factors as the administered agent will patient’s age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of antibody which is in the range of from about 1 pg/kg- to ‘lo -mgykg (body weight of ‘patient),i although a lower for higher dosage may be administered. when providing ICAM5l molecules or their functional derivatives to a patient, iti is preferable to administer such molecules in a dosage which also ranges from about 1 pg/kg to 10 mg/kg (body weight of patient) although a lower or higher dosage may also be administered. As discussed below, the therapeutically effective dose can be lowered if the anti—ICAM-1 antibody is additionally administered with an anti-LFA—l antibody. As used herein, one compound is said to be additionally administered with a second compound when the administration of the two compounds is in such proximity of time that both compounds can be detected at the same time in the patient’s serum.

Both the antibody capable of binding to ICAM—l and ICAM-1 itself may intravenously, intramuscularly, when administration be administered to patients subcutaneously, enterally, or parenterally. administering antibody or ICAM-I by continuous infusion, or by single or multiple boluses.

The anti-inflammatory agents of the present invention are intended to be provided to recipient subjects An amount is injection, the may be by in an amount sufficient to suppress inflammation. said to be sufficient to "suppress" inflammation if the dosage, route of administration, etc. of the agent are sufficient to attenuate or prevent inflammation.

Anti-ICAM-1 antibody, or a fragment thereof, may be administered either alone or in or more additional immunosuppressive agents (especially to a recipient of an organ or The administration of such compound(s) may be for when provided combination with one tissue transplant). either a “prophylactic” or “therapeutic” purpose. prophylactically, the immunosuppressive compound(s) are provided in advance of any inflammatory response or symptom (for example, prior to, at, or shortly after) the time of an organ or tissue transplant but in -30 _. advance of any symptoms of organ rejection). The prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent inflammatory response (such as, for example, rejection of a transplanted organ or tissue, etc.). when provided therapeutically, the immunosuppressive compound(s) is provided at (or shortfly after) the onset of a symptom of actual inflammation (such as, for example, organ or tissue rejection). The administration of ‘the compound(s) serves to attenuate any actual inflammation (such as, for example, the rejection of a transplanted organ or tissue).

The anti—inflammatory agents of the present invention may, thus, be provided either prior to the onset of inflammation (so as to suppress an anticipated inflammation) or after the initiation of inflammation. therapeutic A composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount“ if the amount administered is physiologically significant. in a detectable An agent is physiologically significant if its presence results change in the physiology of a recipient patient.

The antibody and [CAM-1 molecules of the present invention can be formulated according to known methods to prepare pharmaceutically . whereby these materials, or their functional combined in with a Suitable vehicles and their formulation, useful compositions, derivatives, are admixture pharmaceutically acceptable carrier vehicle. inclusive of other human proteins, e.g., described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). In order to form a pharmaceutically acceptable suitable for administration, such compositions will contain an effective amount of anti—ICAM antibody or ICAM-l molecule, or their functional derivatives, together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the release preparations may be achieved human serum albumin, are composition effective duration of action. Control through the use of polymers to complex or absorb anti—ICAM-1 antibody -31_ or ICAM~l, or their functional derivatives. The controlled delivery may be exercised by selecting appropriate polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinyl- acetate, methylcellulose, sulfate) and the concentration of macromolecules as_well as the methods macromolecules (for example carboxymethylcellulose, gr protamine, of incorporation in order to control release. Another possible method to control the duration of action by controlled to incorporate anti~ICAM—l. antibody or ICAM-1 molecules, or their functional derivatives, into particles of a polymeric material such as release preparations is polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, For example, by coacervation for example, poly- respectively, or in colloidal drug techniques or by interfacial polymerization, hydroxymethylcellulose or gelatine~microcapsules and (methylmethacylate) microcapsules, delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in nmcroemulsions.

Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

Having now generally described the invention, readily understood through reference to the following examples which the same will be more are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE 1 Culturing of Mammalian Cells In general, the EBV—transformed and hybridoma cells of the present invention were maintained in RMPI 1640 culture medium, supplemented with 20 mM L-glutamine, 50 pg/ml gentamicin,_and 10% fetal bovine (or Cells were cultured at 37‘C in a 5% C02, 95% fetal calf) sera. air humidity atmosphere. _32_ To establish Epstein-Barr virus (EBV) transformants, 105 T cell depleted peripheral blood mononuclear cells/ml in RPMI 1640 medium supplemented with 20% fetal calf_serum (FCS), and 50 pg/ml gentamicin were incubated for 16 hours with EBV—containing supernatant of 895-8 A (Cantrell, lhe above Exper. Med. herein incorporated by then reference is above procedure are EXAMPLE 2 Assays of Cellular Aggregation and Adhesion In order to assess the extent of cellular adhesion, aggregation assays were employed. Cell lines used in such assays were washed two times with RPMI 1640 medium containing _5 mM Hepes buffer (Sigma Chemical Co., St. Louis) and resuspended to a concentration of 2 x 106 cells/ml. Added to flat-bottomed, 96-well microtiter plates (No. 3596; Costar, Cambridge, MA) were 50 ul of appropriate monoclonal antibody supernatant or 50 pl of complete medium with or without purified monoclonal antibodies, 50 pl of complete medium containing 200 ng/ml of the phorbol ester phorbol myristate acetate (PMA)rand 100 pl of cells at a concentration of 2 x 106 cells/ml in complete medium. This yielded a final concentration of 50 ng/ml PMA and 2 x 105 cells/well.

Cells were allowed to settle spontaneously, and the degree of aggrega- tion was scored at various time points. Scores ranged from 0 to 5+, where 0 indicated that essentially no cells were indicated that less than 10% of the cells were ‘in aggregates; 2+ indicated that less than 50% of the cells were aggregated; 3+ indicated that up to 100% of the cells were in small, loose clusters; 4+ iniclusters; 1+ indicated that up to 100%’ of‘ the« cells were aggregated in larger- clusters; and 5+ indicated that 100% of the cells were in large, very compact aggregates. In order to obtain a more quantitative estimate of cellular adhesion, reagents and cells were added to 5 ml polystyrene in the same order as above. Tubes were placed in a rack on a After 1 hour at approximately 200 rpm, 10 pl tubes gyratory shaker at 37'C. of the cell suspension was placed in a hemocytometer and the number of free cells was quantitated. Percent aggregation was determined by the following equation: _ number of free cells % aggregation = 100 x (1— ——————————————————— -- number of input cells The number of input cells in the above formula is the number of cells per ml in a control tube containing only cells and complete medium that had not been incubated. The number of free cells in the above equation equals the number of non-aggregated cells per ml froni experimental tubes. The above procedures were described by Rothlein, R., et al., Q; EXAMPLE 3 LFA~l Dependent Cellular Aggregation Example 2 was line JY.

The qualitative aggregation assay described in performed using the Epstein—Barr transformed cell Upon As a second means of measuring adhesion, the quantitative assay described in Example 2 was used. Cell suspensions were shaken at 200 transferred to a hemocytometer, aggregates were enumerated- In the absence of PMA, 42% (SD = 20%, N = 6) of JY cells were while JY cells incubated under identical conditions with 50 ng/ml of PMA had 87% (SD = 8%, N = 6) of the cells in aggregates. Kinetic studies of aggregation showed that PMA enhanced the rate and magnitude of aggregation at all time points tested (Figure 3). rpm for 2 hours, and cells not in in aggregates after 2 hours, EXAMPLE 4 Inhibition of Aggregation of Cells Using Anti—LFA-1 Monoclonal Antibodies To examine the effects of anti~LFA—l monoclonal antibodies on PMA- induced cellular aggregation, such antibodies were added to cells in accordance with the qualitative aggregation assay of Example 2. The monoclonal antibodies were found to inhibit the formation of aggregates of cells either in the presence or absence of PMA. Both the F(ab’)2 and Fab’ fragments ‘of inonoclonal’ antibodies against the alpha chain of LFA—l were capable oF“inhibiting cellular incubated Contact- T experiment were described by ’Rothlein, R. aggregation. Whereas essentially 100% of cells formed aggregates in the absence of anti-LFA-1 antibody, less than 20% of the cells were found to be in aggregates when antibody was added. The results of this . V EXAMPLE 5 I Cellular Aggregation Requires the LFA-1 Receptor EBV~transformed lymphoblastoid cells were prepared from patients in the manner described in Example 1. Such cells were screened against monoclonal antibodies capable of recognizing LFA—] and the cells were found to be LFA—1 deficient.

The qualitative aggregation assay described in Fxample 2 was employed, using the LFA-1 deficient cells described above. Such cells failed to spontaneously aggregate, even in the presence of PMA.

EXAMPLE 6 The Discovery of ICAM—l The LFA-1 deficient cells of Example 5 were labeled with carboxyfluorescein diacetate (Patarroyp, M. et al., Cell. Immunol.

To determine whether LFA-1 was important only in forming aggregates, or in their maintenance, antibodies capable of binding to LFA-I were added to the preformed aggregates described above. The addition of antibody was found to strongly disrupt the preformed aggregation. Time ' etm all" IJL‘ Exper. ‘Med: " lapse video recording confirmed that addition of the monoclonal anti~ bodies to preformed aggregates began to cause disruption within 2 hours (Table 1). After addition of nwnoclonal antibodies against LFA-I, pseudopodial movenents and changes in shape of individual cells within ’ aggregates continued unchanged. Individual cells gradually disassociated from the periphery of the aggregatei by 8 hours cells were mostly dispersed. By video time lapse, the disruption of preformed aggregates by LFA-l.monoclonal antibodies appeared equivalent to the aggregation process in the absence of lFA—l monoclonal antibody running backwards in time.

TABLE 1 Ability of Anti-LFA-I Monoclonal Antibodies to Disrupt Preformed PMA—Induced JY Cell Aggregates Aqqreqation score Exp. ' 18 h Zha mmb mmb + 4+ l+b + 4+ 1+C + 5+ 1+d Aggregation in the qualitative microtiter plate assay was scored visually. with anti—LFA-1 present throughout the assay period, aggregation was less than 1+.

‘Amount of aggregation just before addition of Mono- clonal antibody at 2 h. bisi/18 + TS1/22.

C151/18. dlSl/22. * EXAMPLE 7 The Requirement of Divalent Ions for LFA—l Dependent Aggregation Example 2) in medium free of calcium or magnesium ions.

JY celjs failed to aggregate (using the assay of The addition of divalent magnesium supported aggregation at concentrations as low as .3 mM. ions, however, were Found to augment the ability of magnesium ions to Addition of calcium ions alone had little effect. Calcium support PMA'induced aggregation. when 1.25 mM calcium ions were added to the medium, magnesium ion concentrations as low as 0.02 millimolar These data show that the LFA~1 and that can synergize with were found to support aggregation. dependent aggregation of cells requires magnesium ions, calcium ions, though insufficient of themselves, magnesium ions to permit aggregation.

EXAMPLE 8 The Isolation of Hybridoma Cells Capable of Expressing Anti-ICAM-1 Monoclonal Antibodies immunized immunization. The immunizations were administered 45, 29, and 4 days before spleen cells were removed from the mice ineorder to produce the desired hybridoma cell lines. On day 3 before the removal of the spleen cells, the mice were given an additional 107 cells in 0.15 ml medium (intravenously).

Isolated spleen-cells from the aboveldescribed animals were fused " with P3X73Ag8.653 myeloma cells at a ratio of 4:1 acchrding to the protocol of Galfre, G. gt_gl., flgtggg gggzsso (1971). Aliquots of the resulting hybridoma cells were introduced ‘into 96-well microtiter plates. The hybridoma supernatants were screened for inhibition of aggregation, and one inhibitory hybridoma (of over 600 wells tested) was cloned and subcloned by limiting dilution. designated RRI/1.1.1 (hereinafter designated "RR1/1‘).

Monoclonal antibody RR!/1 was consistently found to inhibit PHA- stimulated aggregation of the LFA-I expressing cell line JY. The RR1/1 monoclonal antibody inhibited aggregation equivalently, or slightly some monoclonal to the LFA—1 alpha or beta In contrast, control monoclonal antibody against HLA, which This subclone was less than antibodies subunits. is abundantly expressed on JY cells, did not inhibit aggregation. The antigen antibody RR1/l is defined as the intercellular adhesion molecule-1 (ICAM—1). bound by monoclonal EXAMPLE 9 Use of Anti-ICAM-I Monoclonal Antibodies to Qharacterize the ICkM—1 Molecule monoclonal antibody _39_ with 50 id of a 50% suspension of CNBr—activated, glycine-quenched Sepharose Cl—4B for 1 hour at 4'C. immunoprecipitated with 20 ul of a 50% suspension of monoclonal One milliliter of lysate was antibody RA]/I coupled to Sepharose Cl~4B (1 mg/ml) overnight at 4‘C molecular‘ weights of" 50 kd and 25 kd corresponded to the heavy and subjected to electrophoresis reducing SA) or nonreducing conditions (Figure having light chains of immunoglobulins from the monoclonal antibody Sepharose (Figure 5A, lane 3). Variable amounts of other bands in the 25-50 kd weight range were also observed, but were not seen in precipitates from hairy leukemia cells, which yielded only a 90 kd molecular weight band.

The 177 kd alpha subunit and 95 kd beta subunit of LFA-1 were found to migrate differently from ICAM-1 under both reducing (Figure 5A, lane 2) and nonreducing (Figure 58, lane 2) conditions.

In order to determine the effect df monoclonal antibody RR1/1 on PHA-lymphoblast the quantitative described in Example 2 was employed. Thus, T cell blast cells were stimulated for 4 days with PHA, thoroughly washed, then cultured for 6 days in the presence of IL-2 conditioned medium. PHA was found to be internalized during this 6-day culture, and did not contribute to the aggregation assay. In three different assays with different T cell blast preparations, ICAM-1 monoclonal antibodies consistently inhibited aggregation, aggregation assay aggregation (Table 2).

TABLE 2 Inhibition of PMA—Stimulated PHA-Lymphoblast Aggregation by RR1/1 Monoclonal Antibody? % % Expt. PMA MAb Aggregation Inhibitienb 1C — Control 9 -3 + Control *5] ’ ~ 0 + HLA-A,B , . 53 1 -14d + LFA—1 alpha ‘ 31 39 , + ICAM—1 — 31 39 29 - Control 10 -- + Control 78 0 + LFA-1 beta 17 78 F + ICAM-1 50 36 3 7 + Control 70 + HLA-A,B 80 -14 + LFA—3 83 -19 + LFA-I alpha 2 97 + LFA-1 beta 3 96 + ICAM—1 34 51 “Aggregation of PHA—induced lymphoblasts stimulated with 50 ng/ml PMA was quantitated indirectly by microscopically counting the number of nonaggregated cells as described in Example 2. bPercent inhibition relative to cells treated with PMA and X63 monoclonal antibody.- _ CAggregation was measured 1 hr after the simultaneous addition of monoclonal antibody and PMA. Cells were shaken at 175 rpm. dA negative number indicates percent enhancement of aggregation.

°Aggregation was measured 1 hr after the simultaneous addition of monoclonal antibody and PMA. Cells were pelleted at 200 x G for 1 min. incubated at 37'C for 15 min. gently resuspended, and shaken for 45 min. at 100 rpm. fCells were pretreated with PMA for 4 hr 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.

LFA—1 monoclonal antibodies were consistently more inhibitory than ICAM—1e monoclonal antibodies, whereas HLA-A, B and LFA-3 monoclonal antibodies were without-effect. monoclonal antibodies tested, only those capable of bindiag to LFA-1 or ICAM-1 were capable of inhibiting cellular adhesion: EXAMPLE I0 ’ Preparation of?Monoclonal Antibody to ICAM—l Immunization .

A Balb/C mouse was immunized intraperitoneally (i.p ) with 0.5 mls of 2 x 107 JY cells in RPMI nmdium 103 days and 24 days prior to fusion. On day 4 and 3 prior to 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 U937 cells (ATCC CRL-1593) were differentiated by incubating them at x 105/ml in RPMI with 10% Fetal Bovine Serum, 1% glutamine and 50 pg/ml ng/ml phorbol—l2~ myristate acetate (PMA) in a sterile polypropylene container. On the third day of this incubation, one—half of the volume of medium was withdrawn and replaced with fresh complete medium containing PMA. On day 4, cells were removed, washed and prepared for immunization. gentamyin (complete medium) containing 2 Fusion Spleen cells from the immunized mice were fused with P3x63 Ag8-653 Selection for Anti-ICAM-I Positive Cells After one week, 50 pl of supernatant were qualitative aggregation assay of Example 2 using both JY and SKW-3 as Cells from supernatants inhibiting JY cell screened in the aggregating cell lines- aggregation but not SKW-3 were selected and cloned 2 times utilizing limiting dilution.

—These;results»indicate that of thev — 42 - This experiment resulted in the identification and cloning of three hybridoma anti—ICAM—l The antibodies produced by these hybridoma lines were separate lines which produced monoclonal antibodies.

Igéégl 1gc2b,'and Igfl; respectively: “The hybridoma cell line which ‘ EXAMPLE 11 The Expression and Regulation of ICAM—l a radioimmune assay was In order to measure ICAM—l expression, developed. In this assay, purified RR]/1 was iodinated using iodogen to a specific activity of 10 uCi/pg. 96 well plates and treated as described for each experiment. The plates were cooled at 4'C by placing in a cold room for 0.5-1 hr, not immediately on ice. The monolayers were washed 3x with cold complete media and then incubated 30 m at 4°C with 1351 RRI/1. were then washed 3x with complete media. The bound 1251 was released using 0.1 N NaOH and counted. The specific activity of the 125! RR1/l was adjusted using unlabeled RRI/1 to obtain a linear signal over the Endothelial cells were grown in The monolayers range of‘ antigen densities encountered in this study. Non-specific binding was determined in the presence of a thousand fold excess of unlabeled RR1/1 and was subtracted from total binding to yield the specific binding.

ICAM-1 expression, measured using the above described radioimmune assay, is increased on human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) by IL-1, TNF, LPS and IFN gamma (Table 3). Saphenous vein endothelial cells were used in this study to confirm the results from umbilical vein endothelial cells in cultured large vein endothelial cells derived-from adult tissue.

The basal endothelial cells than on umbilical vein endothelial cells. expression of ICAM~l is 2 fold higher on saphenous vein Lxposure of umbilical vein endothelial cell to recombinant IL-1 alpha, IL-1 IL-1 alpha, TNF and LPS were the most potent inducers and IL-1 was less potent on a beta, and TNF gamma increase ICAM-1 expression 10-20 fold. :weight basis and also at saturating concentrations tbrgthe response (Table 3). IL~l beta at 100 ng/ml increased ICAH-1 expression by 9 fold on HUVEC and 7.3 fld on HSVEC with half-maximai at 15 ng/ml. rTNF at 50 ng/ml increased ICAM-1 expression 16 fold on HUVEC and 11 fold on HSVEC‘wi_th ‘half maximal effects at 0.5 ng/ml.

Interferon-gamma produced a-significant increase in ICAM-I expression of 5.2 fold on HUVEC or 3.5 told on HSVEC at 10,000 U/ml. The effect of LPS at 10 ng/ml was similar in magnitude to that of rTNF. increase occuring Pairwise combinations of these mediators resulted in additive or slightly less than additive effects on ICAH—1 expression (Table 3). Crossetitration of rTNF with rIL—I beta or rIFN gamma showed no synergism between these at suboptimal or optimal concentrations.

Since LPS increased ICAM~l expression on endothelial cells at levels sometimes found in culture media, the possibility that the basal ICAM~l expression might be due to LPS was examined. when several serum batchs were tested it was found that low endotoxin serum gave lower ICAM—1 basal expression by 25%. All the results reported here were for endothelial cells grown in low endotoxin serum. the LPS neutralizing antibiotic polymyxin B at 10 pg/ml decreased ICAM— 1 expression only an additional 25% (Table 3). The increase in ICAM-1 expression on treatment with IL-1 or TNF was not ieffected by the presence of’ 10 ng/ml polymyxin B which is consistent with the low endotoxin levels in these preparations (Table 3).

However, inclusion of A Condition (16 hr) control 100 ng/ml rIL-1 beta 50 ng/ml rIL—l alpha 50 ng/ml rTNF alpha ug/ml LPS ng/ml rIFN gamma r1L-1 beta + rTNF rIL—1 beta + LPS r1L-1 beta + r1FN gamma rTNF + LPS rTNF + rIFN gamma LPS + IFN gama polymyxin B (10 ug/ml) polymyxin 8 + rIL-1 polymyxin B + rTNF 1 pg/ml LPS polymyxin B + LPS Upregulation of ICAM—1 ex -,44 _ TABLE 3 HUVEC 603 + 11 633 9910 1 538 9650 i 1500 9530 1 512 nmi3w 1410 13986 i 761 7849 i 601 i 1241 13480 1 1189 10206 i 320 23 5390 1 97 9785 1 389 7598 1 432 510 i 44 ' 9x- - 16x x 16x .2x x 23x 13x 24x 22x 17x x 20x 13x 1.1x Anti—ICAMv1 Monoclonal Antibodies Specitically bound (cpu) .Hsv£c ‘1132 1 31 mmi7% 657 388 4002 1 664 660 10870 1 805 8401 3 390 1272 13238 1 761 10987 1 668 .3x l1.2x 9.2x 3.5x x 10x 7.4x 14x 12x 10x pression on HVEC and HSVEC- HUVEC or HSVEC were seeded into 96 well plates at 1:3 from a confluent monolayer and allowed to grow to confluence. or media for 16 hr and t in quadruplicate.

Cells were then treated with the indicated materials he RIA done as in methods. All points were done EXAMPLE 12 Kinetics of Interleukin 1 and Gamma Interferon Induction of ICAM-1 reference is ‘herein described in Example 1. medium heat-inactivated fetal bovine serum.

To each well was All steps, including induction with specific counts antibody]-[cpm with X63]. reagents, were carried out in quadruplicate.

The effect of interleukin 1 with a half-life for ICAM—l induction of 2 hours was more rapid than that of gamma interferon with a half-life of 3.75 hours (Figure 6). The time course of return to resting levels of ICAM-1 appeared to depend upon the cell cycle or rate of cell In quiescent cells, interleukin 1 and gamma interferon effects [CAM—l growth- are stable for 2-3 days, whereas in log phase cultures, Immunol. expression is near baseline 2 days after the removal of these inducing agents.

The dose response curves for-induction of ICAM-1 by recombinant mouse ‘and human interleukin '1; and “Far recombinante huhad“ oamma interferon, are shown in Figure 7. Gamma interferon and interleukin 1 were found to have similar concentration dependencies with nearly identical effects at 1 ng/ml. interleukin 1 also have similar curves, but‘are much less effective ~ The human and mouse recombinant than human interleukin 1 preparations in inducing ICAM-I expression.

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

Furthermore, tunicamycin, an inhibitor of N—linked glycosylation, only inhibited the interleukin 1 effect by 43%. and mRNA required for interleukin 1 and gamma interferon-stimulated increases in These results indicate that protein synthesis, but not N—linked glycosylation, are ICAM~I expression. »47~- TABLE 4 Effects of Cycloheximide, Actinomycin D, and Tunicamycin on ICAM-1 Induction *by;1t"1 and gamma~1FN on—Human Dermal~Fibroblasts3 Goat Anti—Mouse 1gG Specifically Bound (cpm) Treatment anti-ICAM-1 r .anti—HLA—A,B.C Control (4 hr) 1524 1 140 T 11928 1 600 + cycloheximide 1513 1 210 10678 1 471 + actinomycin D 1590 1 46 12276 1 608 + tunicamycin 1461 1 176 12340 1 940 11 1 (10 U/ml) (4 hr) 4264 1 249 12155 1 510 + cycloheximide 1619 1 381 12676 1 446 + actinomycin D 1613 1 88 12294 1 123 + tunicamycin 3084 1 113 13434 1 661 IFN-7 (10 U/ml) (18 hr) 4659 1 109 23675 1 500 + cycloheximide 1461 1 59 10675 1 800 + actinomycin D 1326 1 186 12089 1 S50 aHuman fibroblasts were grown to'a density of 8 X 104 cells/0.32 cmz well. Treatments were carried out in a final volume of 50 pl containing the indicated reagents. Cycloheximide, actinomycin D, and tunicamycin were added at 20 pg/ml, 10 pH, and 2 ug/ml, respectively, at the same time as the cytokines. All points are means of quadruplicate wells 1 SD.

EXAMPLE 13 The Tissue Distribution of [CAM-1 Histochemical studies were performed on frozen tissue of human organs to determine the distribution of ICAM-1 in thymus, lymph nodes, intestine, skin, kidney, To perform such an analysis, frozen tissue sections (4 pm thick) of normal human tissues were fixed in acetone for 10 minutes and stained with the monoclonal antibody, RRI/1 by an immunoperoxidase technique which employed the avidin—biotin complex method (Vector Laboratories, Burlingame, CA) described by Cerf- Bensussan, N- g1_g1. (J. Immunol. 11922615 (1983)1. After incubation with the antibody, the incubated with biotinylated horse anti—mouse and liver. sections were sequentially IgG and avidin—biotinylated peroxidase _48_ complexes. The sections were finally dipped in a solution containing 3-aminoethyl-carbazole (Aldrich Chemical Co., Inc., Milwaukee, ill) to develop a color reaction. The sections were then fixed in 4% 'Aform>aldehyde""fo"r'S minutes and Iwerelcountéirstaiiied ‘witl; h'éma't'oxylin.~ Controls included sections incubated with unrelated monoclonal antibodies instead of the RRI/1 antibody.

ICAM-1 was found to have a distribution most similar to that of the major histocompatibility complex (MHC) Class II antigens. Most of the blood vessels (both small and large) in all tissues showed staining of endothelial cells with ICAM—l antibody. staining was more intense in the interfollicular (paracortical) areas The vascular endothelial in lymph nodes, tonsils, and Peyer’s patches as compared with vessels in kidney, liver, and normal skin. In the liver, the staining was mostly restricted to sinusoidal lining cells; the hepatocytes and the endothelial cells lining most of the portal veins and arteries were not stained. diffuse dendritic staining pattern was observed- In the thymic medulla, staining of large cells and a In the cortex, the staining pattern was focal and predominantly dendritic- lhymocytes were not stained. In the peripheral lymphoid tissue, the germinal center cells of the secondary lymphoid follicles were intensely stained. lymphoid follicles, the staining pattern was mostly dendritic, with no recognizable staining of lymphocytes. Faint staining of cells in the mantle zone was also observed. In addition, cytoplasmic extensions (interdigitating reticulum cells) and a small number of lymphocytes in the interfollicular or paracortical areas stained with the ICAM-1 binding antibody.

Cells resembling macrophages were stained in the lymph nodes and lamina propria of the small intestine. Fibroblast-like cells (spindle- shaped cells) and dendritic cells scattered in the stroma of most of the organs studied stained with the ICAM—1 binding antibody. No staining was discerned in the Langerhans/indeterminant cells in the In some dendritic cells with epidermis. No staining was observed in smooth muscle tissue.

The staining of epithelial cells was consistently seen in the mucosa of the tonsils. Although hepatocytes, bile duct epithelium, intestinal epithelial cells, and tubular epithelial cells in kidney did not stain L."1 in most circumstances, sections of normal kidney tissue obtained from a nephrectomy specimen with renal cell carcinoma showed staining of many proximal ‘tubular cells for ICAM-1. These tubular epithelial cells also staaneai-owner Aanlianti-THLA-DR’ binding antibody. g T In summary, ICAM—l is expressed on non-hematopoietic cells such as vascular endothelial cells and on hematopoietic cells such as tissue macrophages and mitogen-stimulated T lymphocyte blasts. ICAM-I was found to be expressed in low_« amounts on peripheral blood lymphocytes.

EXAMPLE 14 The Purification of ICAM—l by Monoclonal Antibody Affinity Chromatography General purification scheme (1986), and Dustin, M.L. gt__a1. (J. Imunol. 1;1:245 (1986).

ICAM—l was solubilized from cell membranes by lysing the cells in a non-ionic detergent, Triton X-100, at a near neutral pH. The cell lysate containing solubilized ICAM—l was then passed through pre- columns designed to_remove materials that bind nonspecifically to the column matrix material, and then through the monoclonal antibody column matrix, allowing the ICAM-1 to bind to the antibody. The antibody column was then washed with a series of detergent wash buffers of During these washes ICAM-l remained bound to the antibody matrix, while non-binding and weakly binding contaminants were removed. The bound ICAM-1 was then specifically eluted from the column by applying a detergent buffer of pH 12.5. increasing pH up to pH 11.0.

Purification of monoclonal antibody RR]/1 and covalent couplinq_ to Sepharose CL-4B. « The anti—ICAM-1 monoclonal antibody RR1/l was’ purified from the ascites fluid of hybridoma—bearing mice, or from hybridoma culture supernates by standard techniques of ammonium sulfate precipitation and - 50 _ Immunochem.

Upsala, swédén; using a nmdificatioh of the~method of March ét 31: (Auéi;‘” end-over-end rotation.

The suspension was incubated for 18 hours at 4'C with gentle The supernatant was then monitored for unbound and remaining reactive sites on the 0.05 M. antibody by absorbance at 280 nm, activated Sepharose were saturated by adding glycine to Coupling efficiency was usually greater than 90%.

Deterqent solubilization of membrangs_prepared from human spleen.

All procedures were done at 4'C. Frozen human spleen (200 g fragments) from patients with hairy cell leukemia were thawed on 200 ml Tris-saline (50 m Tris, 0.14 M NaCl, pH 7.4 at 4'C) containing 1 mM phenylmethylsulfonylfluoride (PMSF), 0.2 U/ml aprotinin, and 5 mM iodoacetamide. The tissue was cut into small pieces, and homogenized at 4'C with a Tekmar power homogenizer. The volume was then brought to 300 ml with Tris~saline, and 100 ml of 10% Tween 40 (polyoxyethylene sorbitan monopalmitate) in Tris—saline was added to achieve a final ice in concentration of 2.5% Tween 40.

To prepare membranes, the homogenate was extracted using three strokes of a Dounce or, more preferably, a Teflon Potter Elvejhem homogenizer, and 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 Tris—saline. After centrifugation at 1000 x g for 15 minutes, the supernatants from both extractions were combined and centrifuged at 150,000 x g for 1 hour to pellet the membranes. The membranes resuspending in 209 ml Tris-saline, centrifuged at 150,000 x g for 1 hour. The membrane pellet was resuspended in 200 ml Tris~saline and was homogenized with a motorized until the were washed by homogenizer and Teflon pestle suspension was uniformly turbid. The volume was then brought up to 900 ml with Tris—saline, and N—lauroyl sarcosine was added to a final concentration of 1%. After stirring at 4'C for 30 minutes, insoluble material in the detergent llysate was removed by centrifugationflat'150,000 xyg for 1 hour. Alriton X1100 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-lymphoblastojd cells The EBV-transformed B-lymphoblastoid cell line JY was rown in RPMI- 1640 containing 10% fetal calf sérum (FCS) and 10 nw HEPES to an approximate density of 0.8 — 1.0 x 106 cells/ml. To increase the cell surface expression of lCAM—1, phorbol 12—myristate ]3~acetate (PMA) was added at 25 ng/ml for 8~12 hours before harvesting the cells. Sodium vanadate (50 pH) was also added to the cultures during this time. The cells were pelleted by centrifugation at 500 x g for 10 minutes, and washed twice in Hank’s Balanced Salt Solution (HBSS) by resuspension and centrifugation. The cells culture) were lysed in 50 ml of lysis buffer (0.14 M NaCl, 50 mM Tris pH 8.0, 1% Triton X-100, 0.2 U/ml aprotinin, 1 mM PMSF, S0 uM sodium vanadate) by stirring at 4°C for 530 minutes. Unlysed nuclei and insoluble debris were removed by centrifugation at 10,000 x g for 15 minutes, followed by centrifugation of the supernatant at 150,000 x g for 1 hour, and filtration of the supernatant through Whatman 3mm (approximately 5 g per 5 liters of filter paper.

Affinity chromatography of [CAM-1 for structural studies For large scale purification of ICAH—l to be used in structural studies, a column of 10 ml of RR]/1-Sepharose CL—4B (coupled at 2.5 mg of antibody/ml of gel), and two 10 ml pre-columns of CN8r—activated, glycine-quenched Sepharose CL—4B, and rat—IgG coupled to Sepharose CL- 4B (Zmg/ml) were used. The columns were connected in series, and pre— washed with 10 column volumes of lysis buffer, 10 column volumes of pH 12.5 buffer (50 mM triethylamine, 0.1% Triton X-100, pH 12.5 at 4'C), followed by equilibration with 10 column volumes of lysis buffer. One liter of the detergent lysate of human spleen was loaded at a flow rate of O S~1.0 ml per minute. The two pre—columns were used to remove non- specifically binding material from the lysate before passage through the RR]/I-Sepharose column.

Fractions containing [CAM-1 were identified by SDSe followed by silver staining (Morrissey, Under these conditions, the bulk (a small amount of IgG, contaminant). The fractions containing ICAM-1 were pooled and concentrated approximately 20~fold using Centricon—30 microconcentrators (Amicon, Danvers, MA). The purified 1CAM—l was quantitated by Lowry protein assay of an ethanol-precipitated aliquot of the pool: approximately 500 pg of pure ICAM—l were produced from the 200 g of human spleen.

Approximately 200 pg of purified ICAM-1 was subjected to a second purification by SDS-polyacrylamide gel electrophoresis. The band representing ICAM-1 was visualized by soaking the gel in 1 M KCl. The gel region which contained ICAM-1 was then excised and electroeluted according to the method of Hunkapiller gt_al., Meth. Enzymol. 21:227-236 (1983). The purified protein was greater than 98% pure as judged by SDS-PAGE and silver staining. stage preparative Affinit urification of ICAM-l for functional studies _____1_2. _M_-.s_.._._.____.________.._ ICAM~l for use in functional studies was purified from detergent lysates of JY cells as described above, but on a smaller scale (a 1 ml l7niinute'wi'th"'3;':minimumlof 5‘ i 1 - 53 _ column of RR!/I-Sepharose), and with the following modifications. All solutions contained 50 all sodium vanadate. After washing the column with pH 11.0 buffer containing 0.1% Triton X-100, the column was washed again with if-ivelcuolumn volumes”'of;'thei'is’aiiie' ‘buffer’coeta'i’ning'1'l‘%‘:n- o'ctyl-beta-D-glucopyranoside (octylglucoside) in place of 0.1% Triton X-100. The octylglucoside detergent displaces the‘ Triton X-100 bound to the ICAM-1, and unlike Triton X-100, can be subsequently removed by dialysis. The ICAM-1 was then eluted with pH'12.5 buffer containing 1% octylglucoside in place ofi 0.1% Triton X-100, and was analyzed and concentrated as described above.

EXAMPLE TS Characteristics of Purified [CAM 1 {CAM-1 purified from human spleen migrates in SOS-polyacrylamide gels as a broad band of Mr of 72,000 to 91,000. 1CAM—1 purified from JY cells also migrates as a broad band of Mr of 76,500 to 97,000.

These Mr are within the reported range for ICAM-1 immunoprecipitated Mr=90,000 for JY cells, 114,000 on the from different cell sources: myelomonocytic cell line U937, _a_l_., J. Immunol. 133245 (1935)). This wide range in M, has been attributed to an extensive, but variable degree of glycosylation. The non-glycosylated precursor has a Mr 0'1‘ 55,000 (Dustin g_t__al_.). The protein purified from either JY cells or human spleen retains its antigenic activity as evidenced by its ability to re-bind to the original affinity column, and by immunoprecipitation with RRl/1- Sepharose and SDS-polyacrylamide electrophoresis- To produce peptide fragments of ICAM—l, approximately 200 ug.was reduced with 2 will dithiothreitol/27.’. SDS, followed by alkylation with 5 mM iodoacetic acid. The protein was precipitated with ethanol, and redissolved in 0.1 M NH4C03/0.1 mli CaCl2/0.1% zwittergent 3-14 (Calbiochem), and digested with 1% w/w trypsin at 37'C for 4 hours, followed by an additional digestion with 1% trypsin for 12 hours at The tryptic peptides were purified by reverse‘-"phase HPLC using a 0.4 x 15 cm C4 column (Vydac)- to 60% The peptides were eluted with a linear gradient of 0% acetonitrile in 0.1% trifluoroacetic acid, .54 - Seiected peptides were subjected to sequence analysis on a gas phase microsequenator (Applied Biosystems). The sequence information obtained from this study is shown in Tahln : ’ TABLE 5 K Amino Acid Sequences of [CAM-1 Tryotic Peptides Amino _ Peptide ‘ ~ Acid ‘ — Residue 50a 50b 46a 46b’ Xv 45 K AA J U 0 M1 1 [T/V] A (V/A) E V S L E A L V L 2 F S Q P E F N L G L T L/E 3 L I T A L P P 0 s G L P/(G) 4. T S F A A T L V I P V L P P P V R L E G/‘{ Y G L L N T P v T N/I 7 P ‘:1 P P V Y Q T P (N) 8 T P I I T/I G G C P/V (Q) 9 S F G (G) L - L S K (E) E E (Q) — D E T (D) 11 A s D/P K s L s 12 G/S V V P F F C 13 A T D Q S E D 14 G V W V/L A — Q 15 I K T P 16 S K 17 A 18 P 19 X Q 21 L ( ) = Low confidence sequence.

[ ] = Very Tow confidence sequence.

/ = Indicates ambiguity in the sequence; most probable amino acid is listed first.

— Major peptide.

Minor peptide. -b EXAMPLE 16 Cloning of the ICAM-1 Gene The gene for ICAM—1. may‘ be cloned- using any of’ a ‘variety. of procedures. For example, the amino acid sequence information obtained through the sequencing of the tryptic fragments of {CAM-1 (Table 5) can be used to identify an oligonucleotide sequence which would correspond to the ICAM—l gene. Alternatively, the ICAM-L gene can be cloned using anti—ICAM-1 antibody to detect clones which produce ICAM-1.

Cloning of the qene for ICAM-1 through the use of oligonucleotide probes sequence having the lowest redundancy) capable of encoding the ICAM-1 Using the “codon usage rules" of Lathe, tryptic peptide sequences is identified. or set of oligonucleotides, containing the The oligonucleotide, theoretical "most probable" sequence capable of encoding the ICAM-1 identify the sequence of a complementary of oligonucleotides fragments is used to oligonucleotide or set which is capable of hybridizing to the "most probably" sequence,-or set of sequences. An sequence can be such a complementary oligonucleotide containing employed as a probe to identify and isolate the ICAM—1 gene (Maniatis, T., et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982). ,56_ As described in Section C, supra, it is possible to clone the ICAM~1 gene from eukaryotic DNA preparations suspected of containing this gene. To identify and clone the gene which encodes the ICAM-1 protein, oligonucleotide probes described above. Because _it is likely that there will be only two copies of the gene for ‘ICAM—l in a normal diploid cell, and because it is possible that.the ICAH-1 gene may have large non—transcribed intervening sequences (introns) whose cloning is not desired, it is preferabie to isolate ICAM-I-encoding sequences from a CDNA library prepared from the mRNA of an ICAMproducing cell, rather than from genomic DNA. Suitable DNA, or cDNA preparations are enzymatically cleaved, or randomly sheared, and ligated into recombinant vectors. lhe ability of these recombinant vectors to hybridize to the above~described oligonucleotide probes is then Procedures for hybridization are disclosed, for example, in Maniatis, T., A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982) or in Haymes, B.T., et_al;, flgglgic Acid Hybridization a Practical Approach, IRL Press, Oxford, England (1985). Vectors found capable of such hybridization are then analyzed to determine the extent and nature of the [CAM-1 sequences measured- Molecular Cloning which they contain. Based purely on statistical considerations, a gene such as that which encodes the ICAM-1 molecule could be unambiguously identified (via hybridization screening) using an oligonucleotide probe having only 18 nucleotides. ' Thus, in summary, the actual identification of ICAM~l peptide sequences permits the identification of a theoretical "most probable“ DNA sequence, or a set of such sequences, capable of encoding such a By constructing an oligonucleotide complementary to this sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable“ oligonucleotides), one obtains a DNA molecule (or set of DNA molecules), capable of function- ing as a probe to identify and isolate the [CAM-1 gene.

Using the ICAM—l peptide sequences of Table S,vthe sequence of the "most probable" sequence of an oligonucleotide capable of encoding the AA and J peptides was determined (Tables Oligonucleotides Complementary to these sequences were synthesized and peptide. theoretical and 7, respectively). purified for use as probes to isolate ICAM—l gene sequences. Suitable size-selected CDNA libraries were generated from the poly(A)* RNA of both PMA-induced HL—60 cells and from PS-stimulated umbilical vein 'endothelial cells. 5A°size-selectedgcbflh library w3s5preparedfusing2" TABLE 6 Oligonucieotide Complementary to the Most ProbabTe Nucleotide Sequence Capable of Encoding the ICAM-1 AA Peptide Amino Acid - Most Probable ' '7‘ Residue of ICAM-1 Sequence Encoding CompTenent'ary ICAM-1 AA Peptide AA Peptide Seauence ' 3: 162 Glu G C A T _ G c 163 Leu 0 G - T, A G C 164 Asp G C A T C G 165 Leu C G T A G C 166 Arg C G G C G C 167 Pro C G C G C G 168 Gln C G A T G C 169 Gly G C G C C G 170 Leu _ C G T A G C 171 Glu G C A T G C 172 Leu C G T A G C 173 . Phe T A T A T A 174 Glu G C A T G C 3’ S’ TABLE 6 (continued) Oligonucleotide Complementary to the Most Probable 'Nucleotide Sequence Capable of Encoding the [CAM-I AA Peptide Amino Acid Most Probable *Residue of ICAM-1 Sequence Encoding Complementary ICAM—l AA Peptide AA Pentide , ~Sequence Asn A T ; - A T ‘C G 176 Thr ‘ AA T C G C G 177 Ser U A C G A 3’ 5’ Nucleotide Sequence CapabTe of Encoding_the [CAM-1 J A ,‘e .- 'e‘ ‘ 7 ‘ ' "' ‘* ‘_'fT‘ gi Amino Acid TABLE 7 Oligonucleotide Complementary to the Most Probab1e Moet-Pfobeb1e Peptide Rékidue of ICAM—1 Sequence Encoding C0mpTemenCEry ICAM-1 AA Peptide AA Peptide Sequence 5i 3! 19 Va] 6 C T A G‘ C Thr A. T C G C G 21 Cys T A G C C G 22 Ser T A C G C G 23 Thr A T C G C G 24 Ser T A C G C G Cys T A ‘ G C T A 26 Asp G c A T _ C G 27 Gln C G A T G C 28 Pro C G C G C G 29 Lys A T A T 3 S ,61- First strand CDNA was synthesized using 8 pg of poly(A)* RNA, avian myeloblastosis virus reverse transcriptase (Life Sciences) and an oligo(dT) primer. The DNA—RNA hybrid was digested with RNase H (BRL) and the second strand was synthesized using DNA polymerase ‘I (New England Biolabsl. The product was methylated with EcdaRl methylase (New England Biolabs), blunt end ligated to Eco RI linkers (New England Biolabs), digested with Eco RI and size selected on a low melting point agarose gel. cDNA greater than S00bp were ljgated to Agtl0 which had previously been Eco RI digested and‘dephosphorylated (Stratagene) The product of the ligation was then packaged (Stratagene gold). was carried out at 45° for 1 hour. denatured Filters were containing 5X Denhardt's Prehybridization Hybridization was carried out using 32bp (’5—TlGGGCTGGTCACAG- GAGGTGGAGCAGGTGAC) or 47bp (S'—GAGGTGlTCTCAAACAGCTCCAGGCCCTGG GGCCGCAGGTCCAGCTC) anti-sense oligonucleotides based, in the manner discussed above, on the ICAM—l tryptic peptides J and AA, respectively (Table 6 and 7) (Lathe, R., J. Mdiec. 8iol., 1_g3:1-12 (1985)).

Oligonucleotides were end labeled with 7-(3ZP)ATP using T4 polynucleotide kinase and conditions recommended by the manufacturer Following overnight hybridization the filters Phages (New England Biolabsl. were washed twice with 2X SSC/0.1% SDS for 30 minutes at 45'C. were isolated from those plaques which exhibited hybridization, and were purified by successive replating and rescreening. _ 62 _ Cloning of the gene for ICAM-1 throuqh the use of anti-ICAM—l antibody contain a unique cloned DNA or cDNA°fragment.

EXAMPLE 17 Analysis of the CDNA clones and coding regions region was region The longest open untranslated untranslated Identity between the determined from 8 TGA terminating triplet at position 1653. translated amino acid sequence and sequences different tryptic peptides totaling 91 amino acids (underlined in confirmed that authentic ICAM-1 CDNA clonés had been The amino acid sequences of ICAM—1 tryptic‘peptides are figure 8) isolated. shown in Table 8.

TABLE 8 Amino Acid Sequences of lCAM—1 Tryptic Peptides Peptide Residues Seguence J 14-29 X G S V L V T C S T S C 0 Q PVK U 30-39 L L G I E T P L (P) (K) -85 (T) F L T V Y X T X 89-95 V E L A P L P AA 161-182 X E L D L R P Q G L E -— L F E X T S A P X Q L K 232-246 L N P T V T Y G X D S F S A K -295 S F P A P N V (T(I) L X K P Q (V/L) —— Indicates that the sequence continues on the next line.

Underlined sequences were used to prepare oligonucleotide probes. hydrophilic followed by a 24 residue hydrophobic putative transmembrane _ 64 _ domain. The transmembrane domain is immediately followed by several charged residues contained within a 27' residue putative Cytoplasmic domain. a heavily glycosylated but otherwise typical integral membrane protein. depending on cell EXAMPLE 18 ICAM—1 is an Integrin—Binding Member of the Immunoglobulin Supergene family performed using the Acid Alignment of ICAM—1 internal repeats was Microgenie protein alignment program lflueen, C., et al., Nucl.

Four protein sequence databases, maintained by the National Biomedical et al., Research Foundation, were searched for protein sequence similarities using the FASTP program of Williams and Pearson (Lipman, D.J., gt_a1;, Science gg1:1435-1439 (1985)).

Since ICAM—1 is a ligand of an integrin, it was unexpected that it would be a member of the immunoglobulin supergene family. However, inspection of the ICAM—1 sequence shows that it fulfills all criteria - 65 _ proposed for membership in the immunoglobulin supergene family. These criteria are discussed in turn below.

The entire extracellular domain of ICAM—l homologous limmunoglobulin-like domains which are lshowm aligned in Figure 9A. Domains 1-4 are ‘88, 97, 99, and 99 residues long, respectively and thus are of typical domain 5 is truncated within 68 residues. Searches of the NBRF‘data base using the is constructed from 5 lg domain ‘size; FASTP program revealed significant homologies with members of the immunoglobulin supergene family including IgM and IgG C domains, T cell receptor a subunit variable domain,iand alpha 1 beta glycoprotein (Fig. 98—D).

Using the above information, the amino acid sequence of ICAM—1 was members of the amino acid other compared with the sequences of immununoglobulin supergene family. lhree types of Ig superfamily domains, V, Cl, and C2 have been differentiated. Both V and C domains are constructed from 2 fi—sheets linked together by the intradomain disulfide bond; V domains contain 9 anti~parallel B—strands while C domains have 7. Constant domains were divided into the Cl— and C2- sets based on characteristic residues shown in Figure 9A. The C1—set includes proteins involved in antigen recognition- The C2—set includes several Fc receptors and proteins involved in call adhesion including CD2, LFA-3, MAG, and NCAM. ICAM-1 domains were found to be most strongly homologous with domains of the C2—set placing ICAll-1 this similarity to conserved residues in C2 than £1 domains as shown for fl- strands B-F in Figure 9. Also, ICAM~l domains aligned much better with fl—strands A and G of C2 domains than with these strands in V and C1 domains, allowing good alignments across the entire C2 domain strength.

Alignments with C2 domains from NCAM, MAG, and alpha I-5 glycoprotein are shown in Figures 98 and 9C; identity ranged from 28 to 33%.

Alignments with a ‘T cell receptor Va 27% identity and IgM C domain 3 34% identity are also shown (Figures 98, 90).

One of the most important characteristics of immunoglobulin domains is the disulfide-bonded cysteines bridging the B and F 5 strands which in this set; is reflected in stronger stabilizes the fl sheet sandwich; in ICAM-I the cysteines are conserved in all cases except in strand f of domain 4 where a leucine is found which may face into the sandwich and stabilize the contact as proposed for some other V? andl‘C2-sets domains;" The distance between the cysteines (43, 50, 52, and 37 residues) is as described For the C2-set. vein endothelial cell cultures by immunoaffinity Chromatography as described above. Acetone precipitated lCAM—l was resuspended in sample buffer (Laemmli, U.K., fiatggg ggZ:680-685 (1970)) with 0.25% 2—mer« captoethanol or 25 mm iodoacetamide and brought to 100°C for 5 min.

The samples were subjected to SDS-PAGE 4670 and silver staining 4613.

Endothelial cell ICAM—l had an apparent Mr of 100 Kd under reducing conditions and 96 Kd under non-reducing conditions strongly suggesting the presence of intrachain disulfides in native ICAM—l.

Use of the primary sequence to predict secondary structure (Chou, P.Y., et al., Biochem. _l_3;:211-245 (1974)) showed the 7 expected 5- strands in each ICAM—1 domain, labeled a-g in Figure 9A upper, exactly fulfilling the prediction for ah) domain and corresponding to the positions of strands A—H (Figure 9A, lower). Domain 5 lacks the A and C strands but since these form edges of the sheets the sheets could still form, perhaps with strand 0 taking the place of strand C as proposed for some other C2 domains; and the characteristic disulfide bond between the 8 and F strands would be unaffected. Thus, the criteria for domain size, sequence homology, conservedcysteines forming the putative intradomain disulfide bond, presence of’ disulfide bonds, and predicted fl sheet structure are all met for inclusion of ICAM-1 in the immunoglobulin iimnunoglobul in in‘ immunoglobulins supergene family. <'-‘ ICAM-1 was found to be most strongly homologous with the NCAM and MAG glycoproteins of the C2 set. This is of particular interest since both NCAM and MAG mediate cell-cell adhesion. NCAM is important in regulated surface expression of- NCAM ‘and. HAG during nervous ‘system formation and myelinatior. extracellular region, Domain-by domain comparisons ‘..- gly, Ann. Rev. comprise one integrin subfamily. A The other two subfamilies mediate vcell-matrix interactions and recognize the sequence RGO within their complement component C3 which shows no immunoglobulin—like features and Leukocyte Typing shown in Table 9.

TABLE 9 Peptides Within the ICAM—l Sequence Possibly Recognized by LFA-1 -L-R-G—E-K-E-Lv -R~G—E-K—E-L-K-R-E-P- ~L-R-G-E-K-E-L-K-R-E-P~A—V-G-E-P-A-E- -P-R-G-G-S- —P-G-N—N—R-K- -Q—E-D-S-Q-P-M- —T-P-E-R-V-E-L-A-P—L-P-S- —R-R~D-H—H—G—A—N—F—S— —D—L~R~P~QvGeL—E- F ._6g _ is the first example of a member cf the immunoglobulin Although both of these ICAM-1 supergene family which binds to an integrin. families play an important role in cell adhesion, interaétion between? ' Furthermore, purified MAG- heterophilic interaction with a receptor The important cells enveloping axons during myelin sheath formation might be due to interaction with a distinct receptor, or due to homophilic MAG-MAG interactions. The homology with NCAM and the frequent occurrence of domain—domain interactions within the‘immunoglobulin supergene family raises the possibility that ICAM-1 could engage‘ in homophilic interactions as well as ICAM—1—LFA—1 heterophilic interactions.

However, binding of B lymphoblast cells which co—express similar densities of LFA~l and ICAM-1 to ICAM-1 in artificial or cellular inhibited by pretreatment -of the B lymphoblast with LFA—l MAb, while adherence is unaffected by B lymphoblast pretreatment with ICAM~l MAb. Pretreatment of the monolayer with [CAM-1 Mab completely abolishes binding (Dustin, M.L-, monolayers can be completely interactions occur at all, they must be much weaker than heterophiiic interaction with LFA-1.

The possibility that the leukocyte integrins recognize ligands in a fundamentally different way is consistent with the presence of a 180' This is in agreement with the cell EXAMPLE 19 Southern and Northern Blots Southern blots were performed using a 5 ug of genomic DNA extracted from three cell lines: BL2, a Burkitt lymphoma cell line (a gift from Dr. Gilbert Lenoir); JY and Er~LCL, EBV transformed B-lymphoblastoid cell lines.

The DNAs were digested with 5X the manufacturers recommended quantity of 8am I11 and Eco RI endonucleases (New England Biolabs).

Following electrophoresis through a 0.8% agarose gel, the DNAs were transferred to a nylon membrane (Zeta Probe, BioRad). The filter was prehybridized and hybridized following standard procedures using ICAM CDNA from HL-60 ‘labeled with a—(32P)d XTP’s by random priming Northern blots were performed using 20 pg of RNA:;was denatured and (Boehringer Mannheim). total RNA or 6 pg of poly(A)+ RNA. through a 1% electrotransferred to Zeta Probe. hybridized as described previously (Staunton, D.E-, _6_-;36'9s-3701' (1987)) using’ the HL-60 coin phobia M32941‘.-;bei‘ed oligonucleotide probes (described above). 9 The Southern blots using the 3 kb CDNA probe and genomic DNA digested with Bam H1 and Eco RI showed single predominant hybridizing fragments of 20 and 8 kb, C suggesting that most of the'coding information is present within 8 kb.

In blots of 3 different cell lines there is no evidence of restriction electrophoresed agarose—formaldehyde gel and Filters were prehybridized and et al. respectively, suggesting a single gene and fragment polymorphism.

EXAMPLE 20 Expression of the [CAM-1 Gene An “expression vector” is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or CDNA) molecule which has been cloned into the vector and ofi thereby producing a polypeptide or protein.

Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expression then the appropriate host cell prokaryotic cell capable of expressing the cloned Similarly, if a eukaryotic expression vector is employed, then the host cell would be any eukaryotic expressing the cloned sequences. Importantly, since eukaryotic DNA may intervening sequences, and since such sequences cannot be it is preferable to employ in order to vector is employed, would be any SEQUERCES . cell capable of appropriate contain correctly processed in prokaryotic cells, cDNA from a cell which is capable of expressing ICAM-1 produce a prokaryotic genomic expression vector library. for preparing CDNA and for producing a genomic library are disclosed by Maniatis, T., et al. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).

Procedures (Molecular Cloning: Embo J. -72, The above—described expression vector genomic library is used to create a bank of host cells (each of which contains one member of the library). The expression vector may be introduced into the host cell by any Zof; a (variety. of ‘means l(i.eL, ‘transformation, gmansfection, protoplast fusion, electroporation, etc.). The bank an’ expression vector-containing cells is clonally propagated, and its members are individually assayed (using an immunoassay) to determine whether they produce a protein capable of binding to anti—ICAM-1 antibody.

V The expression vectors -of those cells 'hhich produce a protein capable of binding to anti-ICAM+l antibody are then further analyzed to determine whether they express (and thus contain) the entire ICAM—1 gene, whether they express (and contain) only a fragment of the ICAM~l gene whose product, whether (and contain} a though immunologically related to lCAM—1, is not [CAM-1. gene, or they express Although such an analysis may be performed by any convenient means, it is preferable to determine the nucleotide sequence of the DNA or CONA fragment which had been cloned into the expression vector. Such nucleotide sequences are then examined to determine whether they are capable of encoding acid the tryptic amino sequence as polypeptides having the same digestion fragments of ICAM—1 (Table 5).

An expression vector which contains a DNA or cDNA nwlecule which encodes the ICAM—l gene may, thus, be recognized by: (i) the ability to direct the expression of a protein which is capable of binding to anti—ICAM-1 antibody; and (ii) the presence of a nucleotide sequence which is capable of encoding each of the tryptic fragments of ICAM-1.

The cloned DNA molecule of such an expression vector may be removed from the expression vector and isolated in pure form.

EXAMPLE 21 Functional Activities of Purified ICAM-I In cells, ICAM~l normally functions as a surface protein associated Therefore, the function of purified [CAM-1 was lipid with the cell membrane. tested after the molecule was reconstituted ihto artificial ' octylglucoside as described above was reconstituted into éesiclest membranes (liposomes, or vesicles) by dissolving the detergent—solubilized lipids, protein in followed by the removal of the detergent by dialysis. the ICAM-1 containing vesicles were fused to glass Zoverslips or plastic culture wells to allow the detection of cells binding to the protein.

Preparation of planar membrahes and plastic-bound vesicles Vesicles were prepared by the method of’ Gay et al. (J. Immunol.

Briefly, were dissolved lipid mixture was dried to a thin film while rotating under a stream of nitrogen gas, and was then lyophilized for 1 hour to remove all traces of chloroform. lhe lipid film was then dissolved in 1% octylgluco— side/0.14 M NaCl/20 mM Tris (pH 7.2) phosphatidylcholine of 0.1 mM. or human glycophorin (Sigma Chemical Co., St. to a final concentration of Approximately 10 ug of purified ICAM-1, Louis, MO) as a control membrane glycoprotein, was added to each ml of dissolved lipids. The then dialyzed at 4’C against 3 pH 7.2, protein—lipid-detergent solution was changes of 200 volumes of 20 M lris/0.14 M NaCl, change of HBSS. and one Proc.

Glass coverslips (11 mm in diameter) the wells were remove unbound vesicles. The planar membrane surface was never exposed (‘ to air. v {CAM-l purified from JY cells and eluted in the detergent for planar membranes.

Cell adhesion assays Cell vesicles were both done in essentially the same way, except that the adhesion assays using planar membranes or plastic—bound cell numbers and volumes for PBV assays were reduced to one~fifth that bound vesicles were pretreated with 20 pg/ml of purified antibody in "loin diéiyzéd arts, ‘am can and iigci "were added ,75 _ RPM!-1640/10% FCS at 4'C for 30 minutes, followed by 4 washes to remove unbound antibody. In experiments on the effects of divalent cations on cell binding, the cells were washed once with Ca2*, Mg2*—free HBSS plus " to yiéir "indicated concentrations. In all experiments, cells and planar membranes or PBV were pre-equilibrated at the appropriate temperature (4‘C, 22‘C, or 37'C) in the appropriate assay buffer. ’ To measure cell binding to purified [CAM-1} 51Cr~labeled cells (5 x 105 EBV-transformants in -planar ‘membrane 105 EBV- transformants or SKN-3 cells, 2 x 105 Con—A blasts in PBV assays) were centrifuged for 2 minutes at 25 x g onto planar‘ membranes or PBV, After incubation, unbound cells were removed by eight cycles of filling and assays; 1 x followed by incubation at 4'C, Z2‘C, or 37'C for one hour. aspiration with buffer pre—equilibrated to the appropriate temperature.

Bound cells were quantitated by solubilization of well contents with 0.1 N NaOH/1% Triton X-100 and counting cell binding was determined by dividing cpm from bound cells by input in a gamma counter. Percent input cpm were planar membrane assays, cell~associated cpm. In corrected for the ratio of the surface area of coverslips compared to the surface area of the culture wells- In these assays, EBV-transformed 8-lymphoblastoid cells, SKw~3 T- lymphoma cells, and Con-A lllymphoblasts bound specifically to ICAM-1 in artificial membranes (Figures 11 and 12}. The binding was specific since the cells bound very poorly to control vesicles containing equivalent amounts of another human cell surface glycoprotein, glycophorin. LFA-1 EBV- transformants and Con~A blasts bound, while their LFA-1 negative counterparts failed to bind to any significant extent, demonstrating that the binding was dependent on the presence of LFA~1 on the cells.

Both the specificity of cell binding and the dependence on_cellular LFA-1 were confirmed in monoclonal antibody blocking experiments (Figure 13). The binding of JY cells could be inhibited by 97% when the ICAM-1—containing PBV were pretreated with anti-ICAM—l monoclonal antibody RR]/1. Pretreatment of the cells with the same antibody had planar membranes or Furthermore, positive U‘‘\ ,76- little effect. Conversely, the anti-LFA—l monoclonal antibody TS]/18 inhibited binding by 96%, but only when the cells, but not the PBV, were pretreated. A control antibody TS2/9 reactive with LFA-3 (a different’ lymphocyte’ surface antigen) had‘ no significant iinhibitory effect when either cells or PBV were pretreated. This experiment demonstrates that ICAM-I itself in the artificialgmembranes, some minor contaminant, mediates the observed cellular adhesion and that the adhesion is dependent on LFA-1 on the binding cell.

The binding of cells ‘to ICAM-1 in artificial membranes displayed two other characteristiés of the LFA-I dependent adhesion and not also system: temperature dependence and a requirement for divalent cations.

As shown in Figure 14, Con~A blasts bound to ICAM‘1 in PBV most effec- tively at 37°C, partially at 22°C, and very poorly at 4°C. As shown in Figure 15, the binding was completely dependent on the presence of divalent cations. At physiological concentrations, Mg2*‘ alone was sufficient for maximal cell binding, while Ca2+ alone produced very low levels of binding. However, Hg2+ at one-tenth of the normal concentra— tion combined with Ca2* was synergistic and produced maximal binding. binding to purified ICAM—1 inhibition with cation In summary, the specificity of cell into artificial membranes. the specific monoclonal antibodies, and the requirements demonstrate that ICAM-1 is a specific ligand for the LFA- ~dependent adhesion.system. incorporated temperature and divalent EXAMPLE 22 Expression of ICAM—l and HLA—DR in Allergic and Toxic Patch Test Reactions Skin biopsies of five normal individuals were studied for their expression of ICAM-1 and HLA-DR. It was found that while the endothelial cells in some blood vessels usually expressed ICAM—l, there was no ICAM-1 expressed on keratinocytes from normal skin. No staining for HLA-DR on any keratinocyte from normal skin biopsies was observed.

The kinetics of expression of ICAM—1 and class 31 antigens were then studied on cells in biopsies of allergic and toxic skin lesions. It was found that one—half of the six subjects studied had keratinocytes which expressed ICAM-1, four hours after application of the hapten lIable ]0)Z“llhere was an increase in the percentage of individuals” expressing ICAM-1 on their keratinocytes with time of efibosure to 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 point a proportion of keratinocytes in all biopsies stained positively for ICAM—1. At 72 hours (24 hours after the hapten was removed), seven of the eight subjects still had ICAM-1 expressed on their keratinyocytes while the expression of ICAM—l on one subject waned between 48 and 72 hours.

TABLE 10 Kinetics of Induction of ICAM~l and HLA~DR on Keratinocytes from Allergic Patch Test Biopsies Time After Patch No. of ICAM-1 HLA-DR ICAM-I& Application (h) Biopsies Only Only HLA-DR Normal Skin 5 O 0 0 X Allergic Patch Test 4 6 33 0 0 8 9 3 0 0 24 8 7 0 0 48b 3 5 0 3 72 8 6 O 1 asamples were considered as positive if at least small clusters of keratinocytes were stained- bAll patches were removed at this time point.: ,78, Histologically, the staining pattern for ICAM-I on keratinocytes from biopsies taken four hours after application of the hapten was usually in small clusters. At 48 hours, ICAM-1 was expressed on the surface of the majority of the keratinocytes, no differenge being seen between the center and periphery of the lesion. The intensity of the staining decreased as the keratinocytes approached the stratum corneum.

This was found in biopsies taken from both the center and the periphery of the lesions. Also at this time point, the patch test was positive and vesicles). [CAM-1 different haptens applied on (infiltration, erythema No difference in was observed when In addition to keratinocytes, ICAM-I was also expression were sensitive individuals. expressed on some mononuclear cells and endothelial cells at the site of the lesion.

HLA~DR on in the allergic skin lesions was less frequent than that of [CAM-I. studied had lesions with keratinocytes that stained positively for HLA- DR up to 24 hours after the application of the hapten- In fact, only four biopsy samples had keratinocytes that expressed HLA—DR and no biopsy had keratinocytes that was positive for HLA~DR and not ICAM-1 (Table 10).

In contrast to the allergic patch test lesion, the toxic patch test lesion induced with sulfate had keratinocytes that displayed little iflany ICAM~l on their surfaces at all time points tested (Table II). In fact, at 48 hours after the patch application, which was the optimum time ICAM~1 expression in the allergic patch test subjects, only one of the 14 toxic patch test subjects had keratinocytes expressing ICAM—I in their lesions. Also in contrast to the allergic patch test biopsies, there was no HLA—DR expressed on keratinocytes of toxic patch test lesions.

These data indicate that ICAM-1 is imune~based inflammation and not in toxic based and thus the expression of ICAM—l may be used to distinguish between immuno based and toxic based inflammation, such as acute renal failure in kidney transplant patients where it is difficult to deteimine whether failure lhe expression of keratinocytes None of the subjects croton oil or sodium. lauryl point for expressed in inflammation, is due to rejection or nephrotoxicity of the immuno-suppressive therapeutic agent. Renal biopsy and assessment of’ upregulation of IABLE 11 Kinetics of Induction of ICAM~l and Ht;-DR on Keratinocytes from Togic Patch Test Biopsies Time After Patch No. of ICAM—1 HLA-DR ICAM-1& Application (h) Biopsies Only Only HLA—UR 0 0 U 13 0 0 24 3 I O 0 48b 14 1 0 0 72 3 I 0 0 “Samples were considered as positive if at least small clusters of keratinocytes were stained- bAll patches were removed at this time point.

EXAMPLE 23 Expression of ICAM-I and HLA-DR in Benign Cutaneous Diseases Cells from skin biopsies of lesions from patients with various types of inflammatory skin diseases were studied for their expression of ICAM-1 and HLA-DR. A proportion of keratinocytes in biopsies of allergic lichen planus contact eczema, pemphigoid/pemphigus and {CAM-1. staining with a pattern similar to or even stronger than that seen in Consistent with expressed Lichen planus biopsies showed the most intense the 48-hour allergic patch test biopsies (Table F2). results seen in the allergic patch test, the most intensive ICAM-I staining was seen at sites of heary mononuclear cell Furthermore, 8 out of the 11 Lichen planus biopsies positiue for HLA3DR expression on keisiineeytes. 3 » The expression of ICAM-I on keratinocytes from skin biopsies of patients with exanthema and urticaria was less pronounced. out of the seven patients tested with these diseases had keratinocytes that expressed ICAM-1 at the site of the lesion. only found on one patient and this ups in conjunction with ICAM-1.

Endothelial of the infiltrate from all inflamatory expressed ICAM—l to a varying extent. infiltration. tested were Only four HLA-DR expression was Cell tested cells and — a proportion mononuclear the benign skin diseases lABLE 12 Expression of ICAM—1 and HLA—DR on Keratinocytes from Benign Cutaneous Diseases No. of ICAM—l HLA—DR ICAM—l& Diagnosis Cases Only Only HLA-DR Allergic Contact Eczema 5 33 \ 0 2 Lichen Planus ‘ll 3 0 8 Pemphigoid/ Pemphigus 2 2 0 O Exanthema 3 2 0 0 Urticaria 4 I 0 1 Samples were considered as positive if at least small clusters of keratinocytes were stained. e -81, EXAMPLE 24 A.Expression_pf ICAM:1 on Keratinocytes of Psoriatic I l Skin Lesions ' V 7 S e The expression of ICAM—1 in skin biopsies from 5 patients with psoriasis were studied before the initiation and periodically during a course of PUVA treatment. Biopsies were.obtained from 5 patients with classical psoriasis confirmed by< histology. Biopsies were taken sequentially before and during indicated time of PUVA treatment. PUVA was given 3 to 4 times weekly- Biopsies were taken from the periphery of the psoriatic plaques in five patients and, in addition biopsies were taken from clinically normal skin in four of the patients. specimens were frozen and stored in liquid Fresh skin biopsy nitrogen. Six micron cryostat sections were air dried overnight at room temperature, Fixed in acetone for 10 minutes and either stained and stored at ~80‘C until immediately or wrapped in aluminum foil staining.

Staining was accomplished in the following manner. incubated with monoclonal antibodies and stained by a three stage Cancer Res 52:57- Sections were immunoperoxidase method (Stein, H., et. al., Adv. 147, (1984)), using a diaminobenzidine H202, substrate. Tonsils and lymph nodes were used as positive control for anti-ICAM—1 and HLA—DR staining. Tissue stained in the absence of primary antibody were negative controls.

The monoclonal antibodies against HLA—DR were purchased from Becton (Mountainview, anti-ICAM-I Peroxidase-conjugated rabbit anti—mouse 1g and Dickinson California). The monoclonal antibody was R606. peroxidase~conjugated DAKAPATTS, were obtained from Sigma (St. Louis, Mo.).

The results of the study show that the endothelial cells in some blood vessels express ICAM—l in both diseased and normal skin, but the purchased from swine anti-rabbit Ig were Copenhagen, Denmark. Diaminobenzidine-tetrahydrochloride intensity of staining and the number of blood vessels expressing ICAM—l was increased in the psoriatic skin lesions. Moreover, the pattern of expression of ICAM-1 in keratinocytes of untreated psoriatic skin lesions from the five patients varied from only small clusters of cells stéifiing*tb many keratinocytes being stained.

PUVA treatment, the ICAM-1 expression on 2 of the patients (patients 2 and 3) showed marked reduction which preceded or was concurrent with During use course of clinical remission (Table 13). of ICAM-1 expression .during. the PUVA treatment which Patients_l, 4 and 5 had decreases and increases » correlated to clinical remissions and relapses, was no ICAM-1 expression on keratinocytes from normal skin before or after PUVA treatment. This indicates that PUVA does not induce ICAM—1 respectively. There on keratinocytes from normal skin.

Of note was the observation that the density of the mononuclear cell infiltrate correlated with the amount of ICAMAI expression on keratinocytes. This pertained to both a decreased number of mononuclear cells in lesions during PUVA treatment when ICAM-l expression also waned and an increased number of znononuclear cells during PUVA treatment when ICAM-I expression on keratinocytes was more prominent. Endothelial ICAM—l-positive. confined to endothelial cells with no labelling of keratinocytes. cells and dermal mononuclear cells are also In clinically normal skin, ICAM-1 expression was The expression of HLA—DR on keratinocytes was variable. There was no HLA-DR positive biopsy that was notlalso ICAM—1 positive.

In summary, these results show that before treatment, ICAM-1 expression is pronounced on the keratinocytes and correlate to a dense mononuclear cellular infiltrate. During PUVA treatment a pronounced decrease of the ICAM—1 staining is seen to parallel improvement. Histologically the dermal infiltrate also diminished. when a clinical relapse was obvious during treatment, the expression of ICAM-1 on the keratinocytes increased, as well as the density of the seen during the clinical infiltrate. when a clinical remission was there was a concurrent decrease in ICAM-1 staining on the dermal treatment, keratinocytes as well as decrease in the dermal infiltrate. Thus the expression of ICAM—l on keratinocytes corresponded to the density of cinflammatory response.

THLA-DR expression on keratihocytes during PUVA treatment. _83_ the mononuclear cellular infiltrate of the dermis. These data show that clinical response to PUVA treatment resulted in a pronounced expression on keratinocytes ‘is responsible for initiating and maintaining the dermal infiltrate and that PUVA treatment down regulates ICAM-1 which in turn mitigates the dermal infiltrate and the The data also indicates that there was variable The expression of ICAh—l on 'keratinocytes of psorfatic lesions correlates with the clinical severity of the lesion as well as with the size of the dermal infiltrate. Thus ICAM-I plays a central 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. Furthermore, monitoring lCAM—l expression on keratinocytes will be an effective tool for diagnosis and prognosis, as well as evaluating the course of therapy of psoriasis.

TABLE 13 Sequential ICAM-1 Expression by Keratinocytes in Psoriatic Skin Lesions (PS) and Clinically Normal Skin (N) and during PUVA treatment PS PS N 'PS ‘N - PS N PS N + + — ++ - ++ — +++ - 1 day + 1 week + + ~ — - ++ » 4 — 0 2 weeks ++ + — 4— — + 3 weeks ++ * 0 4 weeks ++ + — ++ » * * ~6 weeks — ~ 0 7 weeks (++) (+) +++ — t i weeks (+) — ~ +++ Many positive keratinocytes ++ A proposition of positive keratinocytes + Focal positive keratinocytes (+) Very few scattered positive keratinocytes — No positive staining * Clinical remission Clinical relapse EXAMPLE 25 Expression of [CAM-1 and HLA-DR in Malignant Cutaneous Diseases Unlike lesions from benign cutaneous conditions, the expression of ICAM-1 on keratinocytes from malignant skin lesions was much more variable (Table 14). Of the 23 cutaneous T~ceL1 lymphomas studied, ICAM—1 positive keratinocytes were identified in only 14 cases. There _ 85 - was a tendency for keratinocytes from biopsies of mycosis fungoides lesions to lose their ICAM-1 expression with progression of the disease to more advanced stages. However, ICAM-1 expression was observed on a varying proportion of the mononuclearicell infiltrate from most of the A cutaneous T cell lymphoma lesions. Among the remaining lymphomas studied, four of eight had keratinocytes that expressed ICAM—l. Of the 29' patients with malignant cutaneous diseases examined, 5 had keratinocytes that expressed HLA—DR without expressing ICAM—l (Table 14). ' , TABLE 14 Expression of ICAMvl and HLA—DR on Keratinocytes from Malignant Cutaneous Diseases No. of ICAM—l HLA-DR ICAM—l& Diagnosis Cases Only Only HLAVDR CTCL, MP1 8 la 0 4 CTCL, MFII~III 10 1 2 E ClCL, SS 3 l 0 2 CTCL, Large Cell 2 0 Z 0 CBCL _ 2 0 o 1 Leukemia Cutis 3 1 1 1 Histiocytosis X 1 0 O 0 \ 3Samples were considered as positive if at least small clusters of keratinocytes were stained.

EXAMPLE 26 - Effect of Anti-ICAM—1 Antibodies on the Proliferation of Human Peripheral Blood Mononuclear Cells Human peripheral blood mononuclear cells are induced to proliferate molecules, such as the mitogen, concanavalin A, or the T—cell—binding -86 _ antibody OKT3, cause a non~specific proliferation of peripheral blood mononuclear cells to occur.

Human peripheral blood mononuclear cells are heterogeneous in that ‘they 'are composed ,of :subpopulations of” cells‘ which’ are“ capable are “ recognizing specific antigens. when a peripheral blood inononuclear cell which is capable of recognizing a particulaf specific antigen, subpopulation of encounters the antigen, the proliferation of that mononuclear cell is induced. Tetanus~ toxoid and keyhole limpet antigens which are recognized by hemocyanin are examples ’of subpopulations of peripheral mononuclear cells but are not recognizj by all peripheral mononuclear cells in sensitized individuals- The ability of anti—ICAM-1 monoclonal antibody R6~S—D6 to inhibit proliferative responses of 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 as per the manufacturer’s instructions.

Following collection of the interface, the cells were washed three times with RPMI 1640 medium, and cultured in flat-bottomed 96-well microtiter plates at a concentration of 106 cells/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM glutamine, and gentamicin (50 pg/ml).

Antigen, either the T—cell mitogen, concanavalin A (0.25 pg/ml); the T—cell—binding antibody, 0KT3 (0.001 pg/ml); (10 g/ml) or tetanus toxoid (l:100 dilution from source) were added to cells which were cultured as described above in either the presence or absence of anti-ICAM antibody (R606; final concentration of 5 g/ml).

Cells were cultured for 3.5 days (concanavalin A experiment), 2.5 days (0KT3 experiment), or 5.5 days (keyhole limpet hemocyanin and tetanus toxoid experiments) before the assays were terminated.

Eighteen hours prior to the termination of the assay, 2.5 uCi of 3H- Cellular proliferation was keyhole limpet hemocyanin thymidine was added to the cultures. incorporation of thymidine into DNA by the Incorporated thymidine was assayed by measuring the peripheral blood mononuclear cells. collected and counted in a liquid scintillation counter (Merluzzi gt are shown in Figure 16 (concanavalin A experiment), The results of these experiments Figure 17 (OKT3 Figure 18 (keyhole limpet hemocyanin experiment), experiment), _ Figure 19 (tetanus toxoid experiment). - 5 » It was found that anti-ICAM~l antibody inhibits gproliferative responses to the non-specific T-cell mitogen, ConA; fihe non-specific T- cell associated antigen, OKT-3; and the specific antigens, keyhole limpet hemocyanin and tetanus toxoid, ‘ The inhibition by anti-ICAM-1 antibody was comparable to that of anti—LFA-1 antibody suggesting that ICAM-1 is‘a functional ligand of LFA—l and that ICAM-1 inhibit responses. in ,mononuclear cells. antagonism of will specific defense system EXAMPLE 2? Effect of Anti-lCAM—l Antibody on the Mixed Lymphocyte Reaction ICAM~1 immune The inflammatory disease is necessary for effective cellular LFA—l- immune As discussed above, mediated through of ICAM—l responses or the leukocytes with each other and with endothelial cells. when lymphocytes from two unrelated indivduals are cultured in each blast transformation and cell proliferation of the lymphocytes This lymphocytes to the presence of a second population of lymphocytes, is known as a mixed lymphocyte reaction (MLR), and is analogous to the response of lymphocytes to the addition of mitogens (Immunology The Science of Self-Nonself Discrimination, Klein, J., John Wiley & Sons, NY (1982), pp 453-458). .

Experiments were performed to determine the effect of anti-ICAM on the human MLR. These experiments were Peripheral blood was obtained from normal, interactions during an response dependent cell adhesion. induction during allows for interaction of others presence, of one population of are observed. response, monoclonal antibodies conducted as follows- healthy donors by venipuncture. The blood was collected in heparinized tubes and diluted 1:1 at room temperature with Puck's G (GIBCO) balanced salt solution (BSS). The blood mixture (20 ml) was layered over 15 ml of a Ficoll/Hypaque density gradient (Pharmacia, density = 1.078, room temperature) and centrifuged at 1000 x g foe.2O minutes.

The interface was then collected and washed 3X in Puck’s°G. The cells were counted on a hemacytometer and resuspended in RPMI—l64O culture medium (GIBCO) containing 0.5% of gentamicin, 1 mM t—glutamine (GIBCO) and 5% heat inactivated (55‘c,‘ 30 twin.) human AB sera (Flow Laboratories) (hereafter referred to as RPMI-culture medium).

Mouse anti-ICAM—l (R6—5:D6) was’ used in these experiments. All monoclonal antibodies (prepared from ascites by Jackson ImmunoResearch Laboratories, Boston, MA) were used as purified IgG preparations.

Peripheral blood mononuclear cells (PBMC) were cultured in medium at 6.25 x 105 cells/ml in Linbro round—bottomed microliter plates (#764 Ol3—O5). 1000 R and cultured with the responder cells at the same concentration.

Stimulator cells from a separate donor were irradiated at The total volume per culture was 0.2 ml. Controls included responder cells alone as well as stimulator cells alone. The culture plates were incubated at 37’C in a 5% C02-humidified air atmosphere for 5 days- The wells were pulsed with 0.5 uCi of tritiated thymidine (3HT) (New England Nuclear) for the last 18 hours of culture. In some cases a two-way MLR was performed. The protocol was the same except that the second donor's cells were not inactivated by irradiation.

The cells were harvested onto glass fiber filters using an automated multiple sample harvester (Skatron, Norway). rinsing with water and methanol. The filters were oven dried and counted in Aquasol in a Beckman (LS—380l) liquid scintillation counter. as the Mean CPH 3 standard error of 6 individual cultures.

Table 15 shows that purified anti-ICAM-1 monoclonal antibodies inhibited the MLR significant suppression apparent at 20 ng/ml.

Results are reported dose dependent manner with Purified mouse IgG had little or no suppressive effect. of the MLR by the anti-ICAM-1 monoclonal antibody occurs when the antibody is added within the first hours of cultures (Table I6). « in- a Suppression EABLE 15 Effect of Anti-[CAM-I Antibody on the One-Nay Lymphocyte'eaction Responder Cells‘ Stimulator Cellsb*AntibodyC 3HT Incorporation (CPM) - - ~ . — 445d : 143 - + - 148 t 17 + — « 698 1 72 ____._.____*_.___.__._n‘.~fl...__._~s.__.___.__________w______________ r 4 — 4Z,b26 fr, 1,579 : + mlgG (10.0 pg) 36,882 f 1,823 (14%) + + mIgG ( 0.4 pg) 35,500 : 1,383 (17%) + + mlgG ( 0.02 pg) 42,815 :« 1,246 ( 0%) -.._.__s______“__________~_m___ + + R6~S~D6 (10.0 pg) 8,250 : 520 (81%) + R6—5-D6 ( 0.4 pg) 16,142 1 858 (62%) + R6—5~D6 ( 0.03 pg) 28,844 1 1,780 (32%) a. Responder cells (6.25 X I05/ml) b. Stimulator Cells (6.25 x 105/ml, irradiated at IOOOR) c. Purified Monoclonal Antibody to lCAM—l (R6—5-D6) or purified mouse IgG (mIgG) at final concentrations (ug/ml). d. Mean 1 S.E. of 5~6 cultures, numbers in parentheses indicate percent inhibition of MLR -v90- TABLE 16 Time of Addition of Anti-ICAM-1 R3 sb Additionsc 3HT Incorporation (cam) Time of Addition of‘Medium or Antibody Day 0 ‘ Day 1 Day 2 _______________~____..__.__,_____ medium 205d i 14 476 1 132 247 t 75 i medium 189 1 I6 nde nd + » medium 1,860 : 615 nd nd + + medium 41,063 : 2,940 45,955 1 2,947 50,943 1 3,072 + + R6—5—D6 ,781 : %,293 38,409 1 1,681 47,308 1 2,089 (57%) (16%) (7%) . Responder cells (6.25 x 105 ml) . Stimulator Cells (6.25 x 10 /ml, irradiated at IOOOR) c. Culture Medium or Purified Monoclonal Antibody to ICAM—l at 10 pg/ml were added on day 0 at 24 hour intervals d. Mean 1 S.E. of 4-6 cultures . nd = not done . Percent Inhibition (R6—5-D6) the ability of antibody against ICAM—l to inhibit the MLR shows that ICAM—l monoclonal antibodies have therapeutic utility in ICAM-1 monoclonal also have therapeutic utility in related immune mediated disorders dependent on LFA-1/ICAM-1 regulated cell to cell interactions.

The experiments described here show that the addition of monoclonal antibodies to ICAM-1 inhibit the mixed lymphocyte reaction (MLR) when furthermore, ICAM—l In summary, acute graft rejection. antibodies added during the first 24 hours of the reaction.

Amonocytes can be used as angindicator of"inflammation, _ 91 - becomes upregulated on human peripheral blood monocytes during in xitgg culture. particularly if ICAM—l is expressed on fresh monocytes of individuals with acute or chronic inflammation.

[CAM-1's specificity for activated monocytes and the ability of antibody against ICAM—l to inhibit an MLR suggest that ICAM-1 monoclonal antibodies may have diagnostic and therapeutic potential in acute graft rejection and related immune mediated disorders requiring cell to cell interactions.

EXAMPLE 28 Synergistic Effects of the Combined Administration of AntivICAM-I and Anti—LFA—l Antibodies As shown in Example 27, the MLR is inhibited by anti—ICAM—I antibody. The MLR can also be inhibited by the anti~LFA—1 antibody.

In order to determine whether the combined administration of anti»ICAM— 1 and anti-LFA—l antibodies would have an enhanced; or synergistic effect, an MLR conducted in the presence of various assay (performed as described in Example 27) was concentrations of the two antibodies.

This MLR assay revealed that the combination of anti-ICAM-1 and anti-LFA-1, at concentrations where neither antibody alone dramatically inhibits the MLR, is significantly more potent in inhibiting the MLR response (Table 17). This -result indicates that therapies which additionally involve the administration of anti-[CAM-1 antibody (or fragments thereof) and-anti—LFA~l antibody (of fragments thereof) have the capacity to provide an improved anti—inflammatdry therapy. Such an _ 92 _ improved therapy permits the administration of lower doses of antibody than would otherwise be therapeutically effective, and has importance in circumstances where high concentrations of individqgl antibodies induce an anti-idiotypic response. ‘ Q TABLE I7 ‘ Effect of Various Doses of Anti-ICAM-I . T and (R3.1) Anti-LFA~1 on Mixed Lymphocyte Reaction % Inhibition Concentration (uq/ml) Anti~ICAM>1 (R6~S—U6) Anti—LFA~l 0 .004 .02 .1 .5 2.5 0.0 0 7 31 54 69 70 0.0008 1 7 28 48 62 71 0.004 0 13 30 so 64 72 0.02‘ 29 38 64 75 84 as 0.1 92.5 90 91 92 92 92 0.5 93« 90 90 92 93 91 EXAMPLE 29 Additive Effects of Combined Administration of Sub-optimal Doses Anti—ICAM-1 and Other Immunosuppressive Agents in the MLR As shown in Example 28, the MLR is inhibited by combinations of anti-ICAM-1 and anti-LFA—l antibodies. In order‘to determine whether the combined administration of anti—ICAM—l and other immunosuppressive agents (such as dexamethasone, azathioprine, cyclosporin A or steroids (such as, for example, prednisone, etc.) would also have enhanced effects, MLR assays were performed using sub-optimal concentrations at which the agent alone would be provided to a subject) of R6~5-D6 in conjunction with other intnunosuppressive agents as ,per the protocol in Example 27. o . _ The data indicate that the inhibitory effects of R6—5—D6 are at least additive with the inhibitory effects of suboptimal doses of dexamethasone (Table 18), (Table 20). This implies that anti—ICAM—l antibodies can be effective in lowering the thus Azathioprine (Table 19) and cyclosporin A necessary doses of known immunosuppressants, reducing their toxic side effects. In using an anti ICAM I intibody (or a fragment thereof) to achieve such immunosuppression, it is possible to combine the administration of the antibody (or fragment thereof) with either a single additional immunosuppressive agent, or with a combination of more than one additional immunosuppressive agent.

TABLE 18 Effect of Anti—ICAM—I and Dexamethasone on the Human MLR m “ Inhibitor Incorporation % -Group (ng/mi) _ (CPM) Inhibition - Media ,- ° 156 - Stimuiators (S) - ‘ 101 - Responders (R) - 4,461 — R X S — 34,199 — c__,_-c«_.________.____A- ,,,,, ,4 /_ , _ c cc.“ _, , R x 5 R6 9—D6 (8) 25 224 23 R X S Dex (50) I4 I58 59 .n__________*_,c*,-__,c_,__fl_w__. ____- _._,-/W R X S R6-5~D6 (8) + Dex (50) 7,759 77 y%<7 [[[[ ”“””’”*~”‘—7“’%7”7 ” TABLE 19 Effect of Anti-ICAM—I and Azathioprine on the Human MLR ~ 3HT Inhibitor Incorporation % Groug jnggmii (CPM) Inhibition Media - 78 — Stimuiators (S) - 174 — Responders (R) - 3,419 - R x 5 ~ 49,570 - R x S R606 (8) 44,374 11 ~ R x S Azathioprine (1) 42,710 14 R X S R6—5—D6 (8) + Azathioprine (1) 34,246¢ 31 _,__ __ _, ,, ,, __ r _I,/,, ._ ., , .-_._% , _ 95 _ i TABLE 20 Effect of Anti—ICAM-1 and Cyclosporin A on the Human MLR :‘-~— .9?" , 3H1 Inhibitor Incorporation % Group (nq/ml) (cm) Inhibition Media 7 . « : 37 — Stimulators (S) -— ‘ 206 - Responders (R) -- 987 ~ R x S - ' 31,640 — R x S R6—5—D6 (8) 26,282 17 R x 5 CyA (10) 23,617 25 sWVccs_.cM______________c~c,c.“ms.sm,cscccc R x S R6—5-D6 (8) + CyA (10) 19,204 39 Cyfilfltyclosporin A EXAMPLE 30 _ Effect of Anti—ICAM—1 Antibody in Suppressing the Rejection of Transplanted Allogeneic Organs In order to demonstrate the effect of anti-ICAM~1 antibody in suppressing the rejection of an allogeneic transplanted organ, Cynomolgus monkeys were transplanted with allogeneic kidneys according to the method described by Cosimi et al. (Transplant- Proc. 1;:499—503 (1981)) with the modification that valium and ketamine were used as anesthesia.

Thus, the kidney transplantation was performed essentially as Heterotropic renal allografts were performed in 3-5 kg essentially as described" by Marquet (Marquet gt follows.

Cynomolgus monkeys, al;, Medical Primatology, Part II, Basel, Karger,«p- 125 (1972)) after induction of anesthesia with valium and ketafiine. End-to~side anastomoses of donor renal vessels on a patch of aorta or vena cava were constructed using 7-0 Prolene suture. The donor ureter was spatulated and implanted into the bladder by the extravesical approach i’ (Faguchi, Y1; ét ai.;' in’ oéfissét et ‘al.

Transplantation, Baltimore, Williams & Hilkins, Rena} was evaluated by weekly or biweekly In addition, frequent allograft biopsies were obtained p- 393 (1968)). function serum creatinine determinations. for histopathologic examination and complete*autopsies were performed on all nonsurviving recipients. ‘ In most recipients, bilateral nephrectomy was performed at the time of transplantation and subsequent uremic death was considered the end point of allograft survival. In contralateral when some recipients, unilateral native nephrectomy and ureteral ligation were performed at the time of transplantation. allograft rejection occurred, the ligature on the autologous ureter was then removed resulting in restoration of normal renal function and the opportunity to continue immunologic monitoring of the recipient animal.

Monoclonal antibody R6—5~D6 was administered daily for 12 days starting two days prior to transplant at a dose of 1-2 mg/kg/day. levels tested to rejection. The effect of anti«ICAM—1 antibody on the immune system's rejection of the allogeneic kidneys is shown in Table 21. monitor of creatinine were periodically Serum (eds-), linzaf Advances “int TABLE 21 R606 Activity in Prolonging . - Renal Allograft Survival in ' Prophylactic Protocols in the Cynomolgus Monkeya Days of Survival/ Monkey Dose of R6-5:06 (mg/kg) Post-Treatment Control 1 -, _ ‘ 8 Control 2 — ‘ 11 Control 3 — 11 Control 4 - 10 Control 5 9 Control 6 l0 W15 1.0 20 M19 1.0 7*’ M17 1.0 30 M25 1.5 29 M23 1.0 llc M27 2.0 34 M7 0.5 22 M1] 0.5 26 M0 0.5 22 M8 0.5 26d Monkeys were given R6~5—D6 for 12 consecutive days starting at 2 days prior to transplantation. b Cause of death is unknown. There was evidence of latent malaria- C Died of kidney infarct. d Still living as of August 15, 1988.

The results show that R6-5—D6 was effective in prolonging the lives of monkeys receiving allogenic kidney transplants.

EXAMPLE 3! Effect of Anti—ICAM-l Antibody in Suppressing Acute Rejection of Transplanted Organs Q In order to show that anti-ICAM~1 antibody is effective in an acute model of transplant rejection, R6D6 was also tested in a therapeutic or acute kidney rejection model. —In this model, monkey kidneys were stransplanted (using the protocol described in Example 30) and given perioperatively 15 ng/kg cyclosporin A (CyA) i.m. until stable renal function was achieved. The dose of CyA was then reduced biweekly in 2.5 mg/kg increments until rejection occurred as indicated by a rise in blood creatinine levels. At this point. R6—5—D6 was administered for days and the length of survival was monitored. It is important to note that in this protocol, the dose of CyA remains suboptimal since it does not change once the acute rejection episode occurs. In this model (N=5) with no antibody rescue survive 5-14 days To date, Two of these animals historical controls from the onset of the rejection episode. six animals were tested using R6—5—D6 in this protocol (Table 22). 31 days and MS, 47 days Two animals lived 38 and 55 days Following are still surviving (M12, following the administration of R6~5-D6). initiations of R606 therapy and two animals died from causes other than acute rejection (one animal died of CyA toxicity and the other died while being given R6D6 under‘anesthesia). This model more closely approximates the clinical situation in which R6-S—D6 would be initially used. _ 99 - TABLE 22 R6D6 Activity in Prolonging Renal Allograft Survival in Therapeutic Protocols in the Cynomolgus Monkeya Days 6 Survival/ donkey Day of Rejection Episodeb ‘ Post-Treatment Controlsc 14-98 5-14 M24 -' 41 4 ‘ 38 M21 - 34 g 4d M3 41 55 M9 12 119 M12 37 >31‘ M5 26 >47‘ Monkeys were given 1-2 mg/kg of R6—5-D6 for 10 consecutive days following onset of rejection. b Day at which creatinine levels increased as a result of reduction of CyA dosage and R6«5~D6 therapy started.

C Five animals were tested using the therapeutic protocol described above except that there was no rescue therapy- Days of survival/post treatment represents days of survival once creatinine levels started to rise- d Died while under anesthesia. Creatinine levels were low. 9 Died of CyA toxicity. Creatinine Wevels were low. f Still living as of August 15, 1988.

EXAMPLE 32 Genetic Construction and Expression of Truncated Derivatives of ICAM«l In its‘ natural state, ICAM-1 is a cell membrane—bound protein containing an extracellular region of 5 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic domain. It was desirable to construct functional derivatives of ICAM—1 lacking the transmembrane domain and/or the cytoplasmic domain in that a soluble, secreted Form (.,’7 A gut’, ggg’ strain of E. coli transformed with this construct, designated derivative of protein was in the eukaryotic functional lacking the A truncated [CAM-I transmembrane and cytoplasmic domains, but containing the extracellular region possessing all 5 immunoglobulin—like domains was prepared. A 30 bp mutant oligonucleotide (CTC TCC CCC CGG TTC TAG ATT GTC ATC ATC) was used to transform the codons for amino acids tyrosine (Y) and glutamic acid (E) at positions 452 and 453, respectively, to a phenylalanine (F) and a translational stop codon (TAG). The mutant was isolated by its unique Xba I restriction site, and was designated Y452E/F,TAG.

COS cells were transfected with ¢Three days after To express the mutant protein, three mutuant subclones (#2, #7, and #8). in monkey’ cells after transfection with the £ — 101 — transfection with the three mutant subclones, culture supernates and cell lysate were analysed by immunoprecipitation with anti—ICAM-1 monoclonal antibody RR1/1 and SDS-PAGE. ICAM—l was precipitated from the culture supernates of cells transfected with mutanefisubclones #2”" and #8, but not from detergent lysates of those cells- The molecular weight of ICAM-1 found in the approximately 6 kd relative to the membvane_form of ICAM-1, which is consistent with the size predicted from the mutant DNA. Thus, this functional derivative of TEAM-I isfexcreted as a soluble protein. In contrast, {CAM-1 culture supernates of cells transfected with native ICAM—l, demonstrating that culture supernate was reduced was not immunoprecipitated from control the membrane form of ICAM~l is not shed from Cos cells. futhermore, no ICAM 1 was lysates from negative control mock—transfected cells. immunoprecipitated from either culture supernates or cell The truncated ICAM—l secreted from transfected cells was purified by immunoaffinity chromatography with an ICAM-I specific antibody (R6- ~D6) and tested for functional in the presence of the detergent octylglucoside, activity in a Cell binding assay.

After purification preparations containing native ICAM—l or the truncated, secreted form were diluted to a final concentration of 0.25% octylglucoside (a below the critical micelle of the detergent). These preparations of ICAM—1 were allowed to bind to the surfaces of plastic 96-well plates (Nunt), to produce ICAM—l bound to a After washing out unbound material, approximately 75-80% concentration concentration solid-phase. and 83~88% of SKW~3 cells bearing LFA-l on their surface bound specifically to the native and to the truncated forms of ICAM~1, respectively- These data demonstrate that the secreted, truncated soluble ICAM-1 functional derivative retained«both the immunological reactivity and the ability to mediate ICAM—l dependent adhesion which are characteristic of native ICAM—l. - A functional derivative of ICAM-1 lacking only the cytoplasmic domain was prepared by similar methods. A 25 bp oligonucleotide (TC AGC ACG TAC CTC TAG AAC CGC CA) was used to alter the codon for amino acid 476 (Y) to a TAG translational stop codon: The mutant was I designated V475/TAG. cells transfected with the mutant detected a membrane bound form of ICAM-1 with a molecular weight approximately 3-kd less than native IeAMé1;' cells demonstrated a punctate staining pattern similar t3 naive [CAM-1 expressed on LPS-stimulated human endothelial cells. transfected with the mutant DNA specifically bound to purified LFA-1 on Immunoprecipitation analysis and SDS—PAGE of Cos Finally, cells plastic surfaces in a manner similar to Cos cells transfected with native ICAM-1 DNA (Table 23}. « TABLE 23 Ability of Cells Expressing ICAM»l or a Functional Derivative of ICAM—1 to Bind lFA—l % of Cells Expressing lCAM—I that Bind LFAAI in the Presence of: TRANSFECTION Ng_Antibggy RR1g1 Mock 0 0 Native lCAM—I 20 V475/TAG 20 0 EXAMPLE 33 MAPPING OF ICAM—1 FUNCTIONAL DOMAINS Studies of ICAM-I have revealed that the molecule possesses 7 Five of these domains are extracellular (domain 5 being domain 1 being furthest from the cell one domain is a transmembrane domain, and one domain is In order to determine which domains. closest to the cell surface, surface), cytoplasmic (i.e. lies within the cell). domains contribute to the ability of ICAM—1 to bind. LFA—1, epitope mapping To conduct such’ studies, different deletion mutants are prepared and characterized for their capacity to studies may be used. ‘.103 - bind to LFA-l. Alternatively, the studies may be accomplished using anti-ICAM antibody known to interfere with the capacity of ICAM—l to bind LFA-1. Examples suitable antibody RR1/1 (Rothlein, R. -et .51., J.- (Springer, T.A. gt_a1;, U.S. Patent Application Serial No? 07/250,446), LB—2 (Clark, E.A. et al., In: Leukocyte Typing I (A. Bernard, et al., Eds.), Springer—Verlag pp 339—346_(1984)). or CL203 (Staunton, D.E. gt of such include Deletion mutants of ICAM—1’can‘be created by any of a variety of means. It is, however, preferable to produce such mutants via site or by other recombinant means (such as by directed mutagenesis, constructing ICAM~l expressing gene sequences in which sequences that encode particular protein regions have been deleted. Procedures which may be adapted to produce such mutants are well known in the art.

Using such procedures, three ICAM-I deletion mutants were prepared.

The first nmtant lacks amino acid residues F185 through P284 (i.e.

The second mutant lacks amino acid residues The third mutant deletion of domain 3).

P284 through R451 (i.e. deletion of domains 4 and 5). lacks amino acid residues after Y476 (i.e. deletion of‘ cytoplasmic The results of such studies indicate that domains 1, 2, and 3 ICAM—1 interactions with anti-ICAM—1 domain). are predominantly involved in antibody and LFA~l.

EXAMPLE 34 EFFECT OF MUTATIONS IN ICAM—l ON LFA-1 BINDING The ability of ICAM—l to interact with and bind to LFA-1 is mediated by ICAM-1 amino acid residues which are present in domains 1 of the ICAM-1 molecule (Figures 8, 9 and 10). Such interactions are assisted, however, by contributions from amino acids present in domains 2 and 3 of ICAM-1. Thus, among the preferred functional derivatives of the present invention are soluble fragments of the JCAM—1 molecule which contain domains 1, Z, and 3 of ICAM—1- More preferred are soluble Immunoly 'i1_3_z:127o—1274 ‘r1986)).' R‘a'-"5"' to bind to LFA~l- fragments of the ICAM-l molecule which contain domains 1 and 2 of ICAM- I. Most preferred are soluble fragments of the ICAM—1 molecule which contain domain 1 of ICAM-1. first :I’CAM-{domain are inivolvediin the interaction of I‘EAM—l“ and L-FA-T 1'. Substitutions of these amino acids with other amino a'cids alter the ability of ICAH-I to bind LFA-1. substitutions are shown in Figure_25. such mutations on the ability of the resulting mutant ICAM-I molecule Several amino acid residues within the These amino acid residues and the Figure 25 shows the effects of In F:igures 23, residues are described with reference to the one letter code‘ for amino acids, followed by the position of the residue in the ICAM—l molecule. Thus, for example, "E90" refers to the glutamic acid residue at position 90 of lCAM—1.

Similarly, "E9OV“ refers to the dipeptide composed of the glutamic acid residue at position 90 and the valine residue at position 91. The substitution sequence is indicated to the right of the slash ("/“) mark. The V4, R13, 027, 058, and D6056} residues of ICAM~1 are involved in LFA—l binding.

Replacement of these amino acids altered the capacity of ICAM-I to bind to LFA-1. formation of a mutant [CAM-1 molecule which is less able to bind to LFA-1 (Figure 25). Replacement of the R13 residue of ICAM—l with E leads to the formation of a mutant molecule with substantially less capacity to bind LFA_-1. (Figure 25). Replacement of the 058 residue of ICAM—1 with H leads to the formation of a mutant molecule having a substantially normal capacity to bind LFA—l (Figure 25). of the D605 residues of ICAM—l with KL leads to the formation of a mutant molecule having substantially less capacity to bind LFA-1 (Figure 25).

Glycosylation sites in the second domain are also involved in LFA-1 Replacement of N103 with K, or AISSN with SV, results ICAM—1 molecule which is substantially incapable of binding LFA—l. In contrast, replacement of the glycosylation site N175 with A did not appear to substantially effect the capacity of the mutant ICAM—1 to bind LFA-1.

For example, replacement of V4 with G results in the Replacement binding (Figure 23). in the formation of a mutant — 105 « Mutations in the third ICAM—l domain did not appreciably alter ICAM— 1 - LFA—l binding (Figure 24).

EXAMPLE 35 ' 4* MULTIMERIC FORMS OF [CAM-1 wnu INCREASED BIOLOGICAL HALF—LIFE AFFINITY AND CLEARANCE ABILITY Chimeric molecules are constructed in which domains 1 and 2 of ICAM- 1 are attached to the hinge region‘of the immunoglobulin heavy chain.

Preferred constructs attach the Cjterminus of ICAM-I domain 2 to a segment of the immunoglobulin heavy chain gene just N—terminal to the hinge region, allowing the segmental flexibility conferred by the hinge region. lhe ICAM—l domains 1 and 2 will thus replace the fab fragment of an antibody. Attachment to heavy chains of the lgG class and production of animal cells will result in the production of a chimeric molecule. Production of molecules containing heavy chains derived from IgA or IgM will result in production of molecules of higher multimericy containing from 2 to 12 ICAM~l molecules. Co—expression of J-chain gene in the animal cells producing the ICAM—1 heavy chain chimeric molecules will allow proper assembly of IgA and IgM multimers resulting predominantly in IgA molecules containing 4 to 6 ICAM-I molecules and in the case of IgM containing approximately 10 ICAM—l molecules. These chimeric molecules may have several ahvantages. First, Ig molecules are designed to be long lasting in the circulation and this may improve biological half-life.

Furthermore, the multimeric nature of these engineered molecules will allow them to interact with higher avidity with rhinovirus as well as with cell surface LFA-1, depending on the therapeutic context, and thus greatly decrease the amount of recombinant protein which needs to IgA and IgM are highly glycosylated molecules normally present in secretions in mucosal sites as in the nose. Their highly hydrophilic nature helps to keep bacteria and viruses to which they bind out in the mucosa, preventing attachment be administered to give an effective dose. to cells and preventing crossing of the epithelial cell membrane (J‘! - 106 — Thus, they may have increased therapeutic efficacy. [gm and in particularly IgA are stable in mucosal environments and they may increase the stability of the ICAM-1 constructs. If such an {CAM-1 barrier. functional derivative is administered in the blood stream%'it may aiso G increase biological half-life. ,IgA does not fix complement and thus would be ideal for applications in which this wouldgbe deleterious. If IgG H chain chimerics are ‘desired, it would be possible to mutate regions involved in attachment to complement as well as in interactions with Fc receptors. ¢ EXAMPLE 36 GENERATION OF ICAM—1 MUTANTS gt al., Cell directed mutagenesis.

Briefly, E. coli strain X5127 was transformed with pCD1.8. Single colonies were grown in one ml of Luria Broth (LB) medium (Difco) with 13 pg/ml ampicillin and 8 pg/ml tetracycline until near saturation. 100 pl of the culture was infected with R408 helper phage (Strategene) at a multiplicity of infection (MOI) of 10, and 10 ml of LB medium with ampicillin and tetracycline was added for a 16 hr culture at 37‘C.

Following centrifugation at 10,000 rpm for one minute, and 0.22 pm filtration of the supernatant, the phage suspension was used to infect BW3l3/P3 which was then plated on- LB agar (Difco) plates golonies were picked, E. coli supplemented with ampicillin and tetracycline. grown in 1 ml LB medium with ampicillin and tetracycline to near . — 107‘ saturation and infected with helper phage at MOI of 10. increased to 250 ml and the cells were cultured overnight.

Culture volume was then Single strand DNA was isolated by standard phage extraction.

Transfection A . ~ _ COS cells were seeded into 10 tm tissue culture plates such that they would be 50% confluent by 16-24 hrs. once with TBS and incubated for 4 hrs with 4 ml RPMI containing 10% Nu COS cells were then washed sera (Collaborative) S pg/ml chloroquine, 3 pg of mutant plasmid and 200 pg/ml DEAE~dextran Cells DMSO/PBS followed by PBS and cultured 16 hrs in culture media. sulfate. were then washed wit 10% Culture media was replaced with fresh media and at 48 hrs post transfection (OS cells were suspended by trypsin/EDTA (Gibco) treatment and divided into as 24 well tissue culture plates for HRV , 10 cm plates as well binding- At 72 hrs cells were harvested from 10 cm plates with 5 mM EDTA/HESS and processed for adhesion to LFA-1 coated plastic and immunofluorescence.

LFA—l and HRV binding LFA-1 was purified from SKN-3 lysates by immunoaffinity chromatography on lS2/4 LFA—I mAb Sepahrose and eluted at pH 11.5 in the presence of 2 mM MgClz and 1% octylgucoside. LFA—l (10 pg per 200 pl per 6-cm plate) was bound to bacteriological Petri dishes by diluting octylglucoside to 0.1% in PBS (phosphate buffered saline) with 2 mM MgCl2 and overnight incubation at 4‘C. Plates were blocked with 1% BSA (bovine serum albumin) and stored in PBS containing 2mM MgClg, 0.2% BSA, 0.025% azide, and 50 pg/ml gentamycin.

Cr—labelled COS cells in PBS containing 5% FCS (fetal calf serum), 2 mM MgCl2, 0.025% azide (buffer) were incubated with or without 5 V — 108- pg/ml RRI/1 and R6.S in LFA—1 coated microtiter plates at 2S‘C for 1 hour. Non-adherent cells were removed by 3 washed with buffer.

Adherent cells were eluted by the addition of EDTA to 10 mM and 1- counted. ‘ - e 8 RESULTS Anti—ICAM—1 antibodies such as RR1/l,'R6.5; LB—2, or CL203 have been identified. inhibiting ICAM—1 function, they must be capable of binding to a particular site in the ICAM—l molecule which is also important to the ICAM-1 function. Thus, ICAM-1, and antibodies can bind to If these antibodies -are capable of by preparing the above-described deletion mutants of determining the extent to which the anti—lCAM—l the deletion. it is possible to determine whether the deleted domains are important for function.

ICAM-l domain is predicted to be composed of 5 lg—like C—domains. in binding LFA-1, domain 3 and domains 4 (carboxyl deleted by mutagenesis and tested functionally following expression in CO5 cells.

In addition, the entire cytoplasmic domain was deleted to ascertain its ICAM—1 cytoplasmic domain deletion, Y476/*i bemonstrated no loss of RR1/I, R6.S, LB—2 and CLZO3 reactivity whereas, deletion of domain 3, F185- R4Sl, resulted in a decrease and loss of CL203 reactivity, respectively (Figure 20). Thus, the CL203 epitope appears to be located in domain 4 whereas RR1/1, R6.S and LB—2 appear to be located terminal,domains.

All 3 deletion mutants demonstrate wild type levels of adherence to LFA-1 (Figure 21). Amino acid substitutions in predicted fi—turns in 2 and 3 were also generated and functionally tested following expression in COS cells. The R6.S epitope was thus localized to the sequence EIIIGGA in domain 2 and may also involve E39 in domain 1 whereas RR]/1 and L8-2 are both dependent on R13cin domain 1 (Figure is an integral membrane protein, of which the extracellular To identify and 5 oligonucleotide-directed domains involved terminal) were potential influence on interactions. As expected the in the 2 amino- domains 1, _ 109" ). In addition, RR]/1 binding is decreased by mutations in the sequence D7lGQS. Mutations eliminating N—linked glycosylation sites at N103 and N165 result in decreased RR1/1, R5.S and LB—2, LFA«1 HRV binding. These mutations appear to effect processing sueh that ICAM—I dimers are generated. g Q Other mutations in domain 2 or 3 did not result in altered tFA—I adhesion (Figures 23 and 24). both are involved with binding LFA—l (Figure is).

Thus, LFA—l and HRV binding appears to be a function of the amino terminal Ig-like domain of ICAM-11 [CAM amino terminal domains. V; The amino acids in domain 1, R13 and 060 Figure 26 shows an alignment of ‘r ,

Claims (3)

1. An anti—inflammatory agent for use in the preparation of a pharmaceutical composition to be used together with an immunosuppressive drug chosen preferably from dexamethasone, azathioprine and cyclosporin A for treating inflammation resulting from a response of the specific defence system, said anti—inflammlory agent being chosen from an antibody capable of binding to ICAfl—1; a fragment of an antibody, said fragment being capable of binding.to ICAM-1; ICAM—1; a functional derivative of 1CAM—l; and a_non—iunoglobulin antagonist of ICAM-1. ’

2. An anti-inflamatory agent for use in the preparation of a pharmaceutical composition for treating inflammation resulting from a response of the specific defence system to be used together with a second agent chosen from; an antibody capable of binding to LfA—l; a functional derivative of an antibody, said functional derivative being capable of binding to lFA—1; and a non—immunoglobulin antagonist of LfA—l; said anti—inflammatory agent being chosen from an antibody capable of binding to ICAM—1; a fragment of an antibody, said fragment being capable of binding to ICAM-1; ICAM—l; a functional derivative of ICAM—1; and a non-immunoglobulin antagonist of ICAM—1.

3. A pharmaceutical composition comprising: a) an anti—inflammatory agent selected from the group consisting of: an antibody capable of binding to ICAM~l; a fragment of an antibody, said fragment being capable of binding to ICAM-1; ICAM-1, a functional derivative of ICAM—l; and a non-immunoglobulin antagonist of ICAM—1, and b) at least one imunosuppressive agent selected from the group consisting of: dexamethasone, azathioprine and cyclosporin A. An anti—inflammatory agent for use in the preparation of a pharmaceutically composition to be used together with an immunosuppressive drug substantially as described herein with reference to the Examples and/or the description. An anti—inflammatory agent for use in the preparation of a pharmaceutical composition substantially as described herein with reference to the‘Examples and/or thstpe§qription. A pharmaceutical composition substantially as described herein with reference to the Examples and/or the description. TOMKINS & CO.