WO1993019784A1

WO1993019784A1 - Novel endothelial adhesion molecule for monocytes

Info

Publication number: WO1993019784A1
Application number: PCT/US1993/003153
Authority: WO
Inventors: Leslie M. Mcevoy; Eugene C. Butcher
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 1992-04-07
Filing date: 1993-04-06
Publication date: 1993-10-14
Also published as: AU4025493A

Abstract

Methods and compositions are provided for the modulation of monocyte binding to endothelial cells, particularly during inflammatory episodes. Compositions are provided which bind to one or both of the monocyte surface membrane protein or the endothelial surface membrane protein which are complementary or result in the adhesion of the monocyte to the endothelial cell. The subject compositions can be used in diagnosis or therapy.

Description

NOVEL ENDOTHELIAL ADHESION MOLECULE FOR MONOCYTES

INTRODUCTION

Technical Field

The field of this invention is the modulation of monocyte and endothelium response to inflammation and trafficking of onocytes to sites of inflammation.

Background

An important physiological process for monitoring and treating diseased states involves the migration of leukocytes from the vascular system to the site of injury. Depending upon the nature of the injury, different types of cells may be recruited. Thus, different groups of leukocytes, such as lymphocytes, neutrophils, or monocytes, or combinations thereof, may be involved. In addition, there appears to be variation in the types or subsets of cells which may be recruited to particular tissues, such as mucosa, lymph node, cutaneous, and the like. The need to recruit different types of cells depending upon the nature of the injury appears to be orchestrated by the presence of surface membrane proteins on both leukocytes and endothelial cells associated with the vascular vessels. Some of the surface membrane proteins may be associated with signals which provide for upregulation of the surface membrane protein in response to an agent secreted by the cells at the site of injury.

There has been substantial progress made in identifying a number of proteins associated with binding of lymphocytes to mucosal tissue and peripheral lymph nodes associated with extravasation to a site of injury. The process appears to be a multistep process involving a plurality of proteins on both the lymphocyte and the endothelial cell. While much is understood, there still remains substantial mystery concerning the manner in which the lymphocytes are directed from the vascular system to the site of injury. A similar situation exists with neutrophils. However, the trafficking of monocytes has, for the most part, eluded identification of proteins which are associated with the transport of the monocytes to sites of injury and disease.

Relevant Literature

Jutila et al., Transplantation 48:727-731, 1989; and Jutila et al., "Homing Receptors in Lymphocyte, Neutrophil, and Monocyte Interaction with Endothelial Cells," In Leukocyte Adhesion Molecules: Structure, Function and Regulation. T.A. Springer (ed.), Springer-Verlag, New York; pp. 227-235, 1988, describe the binding of various leukocytes to endothelial cells. Berliner (1990) J. Clin. Invest. 85:1260 report that *low density lipoprotein stimulates monocyte endothelial interactions. The enhancement of adhesion of monocytes to vascular endothelium by interleukin-1 is reported by Bevilacqua et al., ibid. 76:2003. Butcher (1990) Am. J. Pathol. 136:3 describes mechanisms that direct leukocyte traffic. Carlos et al., Blood 77:2266 report the binding of human monocytes to two cytokine-induced adhesive ligands on cultured human endothelial cells: ELAM-2 and VCAM-1. See also Cybulski and Gimbrone (1991) Science 251:788. Gerrity (1981) Am. J. Pathol. 103:181 describes the role of the monocyte in atherogenesis. Lewinsohn et al. (1987) -T. τπmιnoi . 138:4313 describes mechanisms for binding of leukocytes with endothelial cells. McEver (1991) J. Cellular Biochem. 45:156 describes GMP-140 as a receptor for monocytes on activated platelets and endothelium. Territo et al. (1989) Arteriosclerosis 9:824 report that BVLDL pretreatment of endothelial monolayers increases monocyte adhesion.

SUMMARY OF THE INVENTION

Methods and compositions are provided associated with monocyte-endothelial cell binding where the monocyte has a surface membrane protein capable of binding to a reciprocal binding member on endothelial cells. Involvement of monocytes in various situations resulting, from upregulation of an endothelial cell adhesion molecule which binds to monocytes may be modulated using various compounds. The compounds may find use in diagnosis and therapy.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions are provided for modulating and directing entities associated with monocytes binding to sites of inflammation. Surface membrane proteins, receptors for these proteins, and compositions resulting from modifications thereof are employed in controlling interactions between monocytes and endothelium, directing substances to sites of inflammation associated with monocyte binding, and diagnosing the presence of biological components associated with monocyte-endothelial adhesion. The molecules involved are associated with an adhesion regulatory pathway different, at least in part, from the adhesion regulatory pathway for lymphocytes and neutrophils.

The pathway as demonstrated by stimulating in vitro endothelial cells and determining the binding kinetics with monocytes indicates a relatively slow response as compared to lymphocyte binding, generally requiring greater than about 2 hours to reach maximum binding, frequently from about 2 to 10 hours, where binding of monocytes may then continue for at least an additional 24 hours, more frequently up to about 72 hours. This effect can be specifically demonstrated with bEnd3 mouse cells (mouse brain-derived polyoma middle T antigen transformed endothelial cell line) , human umbilical vein endothelial cells (HUVEC) or human aortic endothelial cells (HAEC) . The binding can be demonstrated with cell lines such as WEHI78/24, U937, or other monocytoid line. Since there appears to be substantial complementarity between the human and mouse proteins associated with monocyte binding to endothelial cells, mouse and human cells as the endothelial or monocyte partners are substantially fungible.

The subject compositions are associated with, in the case of endothelial cells, proteins which are up regulated in endothelial cells as a result of stimulation, particularly stimulation as a result of local inflammation. External stimulants in culture may include IL-1, lipopolysaccharide, tumor necrosis factor-α, minimally modified-low density lipoprotein (by minimal modification is intended storage or mild oxidation) , and the like. Also included in the subject invention are receptors which specifically bind to these protein(s) , where the receptors include antibodies, surface membrane proteins, e.g. monocytic surface membrane proteins, fragments thereof, generally of from 8 to 100, usually 16 to 60, amino acids and analogs thereof. Also included are nucleic acid sequences encoding the protein(s) associated with the specific binding of monocytes during this period. In addition, the subject compositions include nucleic acid sequences encoding these proteins and receptors, where the sequence may be cDNA, a genomic sequence, or a synthetic sequence, or combinations thereof where the composition may be the coding sequence by itself, in conjunction with transcriptional regulatory regions, associated with a vector, such as a plasmid or virus, or integrated into a genome, particularly a xenogeneic genome. The manner of preparing the cDNA, genomic gene, expression cassette, cloning and expression vectors and the like are described in the literature.

Antibodies may be obtained by immunizing a mammalian xenogeneic immunocompetent host with endothelial cells which have been stimulated with an appropriate stimulant, as described above, usually at least 2 hours prior to immunization, preferably at least about 4 hours prior to stimulation, and usually not more than about 72 hours, more usually not more than about 48 hours. The immunization will be in accord with conventional techniques, where the cells may be injected subcutaneously, intramuscularly, intraperitoneally, intravascularly, etc. Normally, from about 10⁶ to 10⁸ number of cells will be used, which may be divided up into 1 or more injections, usually not more than about 4 injections. The injections may be with or without adjuvant, e.g. complete or incomplete Freund's adjuvant, Specol, alum, etc. If desired, booster injections may be employed at 2 to 4 week intervals, usually there not being more than about 1 to 3 booster injections. The host may be any xenogeneic host, including urine, rodentia, lagomorpha, ovine, porcine, bovine, etc. The particular host is primarily one of convenience, and where monoclonal antibodies are desired, a sufficient supply of splenocytes may be-obtained. Usually within 3 days after completion of the immunization schedule, the antiserum may be harvested in accordance with conventional ways, to provide polyclonal antisera specific for the surface membrane proteins of the endothelial cells, having significant specificity for the endothelial leukocyte adhesion molecule(s) specifically binding to monocytes (monocyte-ELAM) .

Of particular interest are monoclonal antibodies which are characterized by binding to a surface membrane protein of an endothelial cell, where the endothelial cell may be any mammalian endothelial cell, including both venule and arterial endothelial cells, from any mammalian host, particularly primate, more particularly human, where the antibody is capable of at least partially blocking the binding of a monocyte to the endothelial cell. By partially blocking is intended at least 20 percent of the number of cells which bind under the conditions of the screening for binding are inhibited from binding, preferably at least about 25%, and inhibition may be about 50% or more. These conditions should be at the least stringent temperature, namely 4°C, preferably at 25°C, more preferably at about 37°C. Generally, the antibody will be further characterized by not interfering with binding of lymphocytes and neutrophils to the endothelial cells. Of particular interest are the monoclonal antibodies described in the Experimental section, LM151.7 and 141, cross-reactive antibodies thereof, species analogs thereof, humanized forms thereof, binding fragments thereof, and conjugates thereof. These antibodies are characterized by binding to a protein under Western blot conditions from non-reducing SDS-PAGE gels which have a molecular weight based on standards in the range of about 45-50 kD. The protein is found to be expressed at low levels by unstimulated endothelium, but is substantially up-regulated within a few hours upon activation of endothelium with IL-1, TNF-α, LPS, or MM-LDL. The antibodies are found to block binding by at least 25% at both 4° and 25°C of WEHI78/24 cells to bEnd3 cells.

The subject invention is useful in any species, such as primate, particularly human, domestic animals, e.g. murine, bovine, equine, canine, feline, ovine, porcine, etc. , and any of these species may find application as a source of antibodies.

The ability to inhibit immune system functions is known to be therapeutically useful in treating a variety of diseases such as atherosclerosis, allergies, autoimmune diseases, certain malignancies, arthritis, inflammatory bowel diseases, transplant rejection and reperfusion injury. Some of these diseases are listed below in Table 1.

TABLE 1 EXAMPLES OF DISEASES OR IMMUNOLOGICAL DISORDERS Autoimmune and Related Disorders

Systemic Lupus Erythematosus

Rheumatoid Arthritis

Polyarteritis Nodosa

Polymyositis and Dermatomyositis Progressive Systemic Sclerosis (Diffuse Scleroderma) Glomerulonephritis

Myasthenia Gravis Sjogren's Syndrome Hashimoto's Disease and Graves' Disease Adrenalitis, Hypoparathyroidism, and

Associated Diseases

Pernicious Anemia Diabetes Multiple Sclerosis and Related Demyelinating

Diseases Uveitis Pemphigus and Pemphigoid Cirrhosis and

Other Diseases of the Liver

Ulcerative Colitis

Myocarditis Regional Enteritis

Adult Respiratory Distress Syndrome

Local Manifestations of Drug Reactions (dermatitis, etc.) Inflammation-Associated or Allergic Reaction Patterns of the Skin

Atopic Dermatitis and Infantile Eczema Contact Dermatitis Psoriasis Lichen planus Allergic enteropathies

The Atopic Diseases

Allergic Rhinitis Bronchial Asthma

Transplant Rejection (heart, kidney, lung, liver, pancreatic islet cell, others) Hypersensitivitv or Destructive Responses to Infectious

Agents

Poststreptococcal Diseases (e.g. Cardiac manifestations of rheumatic fever)

Others

Another aspect of the invention is the targeting of therapeutic or diagnostic reagents (radiotoxins, reagents capable of inducing vascular permeability to enhance access of soluble blood-borne macromolecular reagents to surrounding tissues or neoplasms, or radiologic, nuclear magnetic resonance or other imaging reagents) to specific tissues or organs. Reagents are covalently linked, using conventional techniques, to antibodies or other specific bindingmolecule to tissue-specific endothelial cell ligands or molecules, and injected intravenously to localize along the vasculature in the target organ or tissue. Such targeting allows novel imaging approaches to the diagnosis of vascular abnormalities or to the evaluation of the vascularization of malignancies. For example, since tissue- specific endothelial cell ligands may be induced inappropriately by factors produced locally by metastatic cells (for instance, mammary gland tissue induces mucosal endothelial ligands locally, and metastatic breast carcinoma might therefore induce monocyte-specific endothelial molecules as well) imaging reagents injected intravenously might readily identify sites of metastatic neoplasms. This approach to imaging of neoplasms, based on changes in the surface of endothelial cells in the local vasculature, avoids the problem of delivery of macromolecules to extravascular sites. The invention also permits localized targeted delivery of therapeutic agents to selected tissues or organs.

The endothelial cell antigens associated with specific monocyte binding may be obtained in substantially pure form from either natural sources or by recombinant techniques. From natural sources, endothelial cells may be stimulated by any of the agents indicated above, or other stimulating agents, and the cells lysed and passed through an affinity colu n of receptor or monoclonal antibody for the antigen. The protein may then be eluted with an appropriate salt solution or aqueous/organic gradient, e.g., acetonitrile, ethanol, etc., usually in the presence of a low acid concentration, 0.1-1 percent trifluoroacetic acid. The eluted protein may then be further purified by chromatography, electrophoresis, or the like in accordance with conventional ways.

Alternatively, the endothelial cell monocyte-ELAM may be obtained by recombinant techniques. Total RNA may be isolated from stimulated endothelial cells shown to express the targeted protein. Residual DNA may be removed in accordance with conventional techniques and the polyadenylated RNA purified further. cDNA may then be prepared in accordance with conventional techniques using reverse transcription. The cDNA may then introduced into an appropriate cloning system, where the cDNA is fused to a marker, such as β-galactosidase. Fusion proteins may then be screened using the subject antibodies or by employing polyclonal antisera, whereby fused proteins which bind to the antibodies may be isolated and utilized in a variety of ways. The protein encoded by the cDNA may be sequenced to establish at least a partial sequence of the protein. The cDNA encoding the protein binding to the antibodies may be used for further probing of the cDNA library for a complete transcript. Alternatively, the cDNA sequence may be used to probe a genomic library to identify the genomic gene encoding the subject monocyte-ELAM. Instead of using molecules which bind to the monocyte-ELAM, fragments of the monocyte-ELAM may be used to bind to the monocytes to inhibit binding to the endothelial cells.

Thus, soluble forms of the monocyte-ELAM may serve to bind to the cell adhesion molecules of the monocyte and inhibit binding to the monocyte-ELAM, where the soluble form may be a protein or fragment thereof, a carbohydrate, glycoprotein, or other molecule capable of mimicking a portion of the monocyte-ELAM which binds to the monocyte cell adhesion molecule. These proteins may include sequences having the same or substantially the same sequence as the monocyte-ELAM, anti-idiotypes, wherethe anti-idiotype binds to an antibody which binds to the monocyte-ELAM, carbohydrate portions of the monocyte-ELAM which bind to a lectin portion of the monocyte cell adhesion molecule, and the like.

For targeting various molecules to post-capillary venules associated with monocyte binding, specific binding molecules, ligands or antibodies may be employed which bind to the monocyte-ELAM. It is not necessary that the specific binding molecules interfere with the binding of the monocyte cell adhesion molecule to the monocyte-ELAM, all that is required is binding to the monocyte-ELAM. The ligands may include carbohydrates which specifically bind to the monocyte-ELAM. The carbohydrate molecules will mimic the sugar portion of the monocyte cell adhesion molecule which binds to a monocyte-ELAM. The sugar molecule may be totally carbohydrate or may have a peptide of fewer than 50, usually fewer than 30, amino acids.

The peptides of the monocyte-ELAM which are employed for binding to the monocyte cell adhesion molecule will usually be at least about 8 amino acids, more usually at least about 12 amino acids, preferably at least about 16 amino acids, and frequently 20 amino acids or more. Various techniques may be employed to extend the lifetime of the smaller peptides, by using an unnatural amino acid as part of the chain, where the unnatural amino acid does not affect the binding conformation of the peptide, by employing liposomes, by modifying the molecule with stabilizing molecules, such as polyethylene glycol, or the like. The molecules may be administered by any convenient means, particularly parenterally, more particularly intravascularly.

The monocyte-ELAM may be employed for identifying the monocyte cell adhesion molecule. Lysates of monocytes, either from appropriate peripheral blood sources of the host or from monocytoid cell lines may be affinity purified, using the monocyte-ELAM, anti-idiotypes which bind to the idiotope of the antibodies to monocyte-ELAM or binding fragments thereof. As described previously, proteins which bind to the affinity column may then be eluted and screened for binding to stimulated endothelial cells. By labeling the proteins eluted from the column, one can detect their binding to stimulate endothelial cells, where the endothelial cells may be from native tissue, cell lines, or the like. The proteins and fractions comprising binding proteins may then be further purified using the techniques described above. The proteins may also be used for producing antibodies to the proteins for further purification and identification. The protein may then be sequenced and probes prepared having redundancy, as appropriate, for screening a cDNA library and/or genomic library of monocytes for isolating sequences encoding the monocyte cell adhesion molecule.

The monocyte cell adhesion molecule or fragments thereof may then be used to direct a wide variety of moieties to sites of inflammation associated with monocyte binding. Labels may include radioactive isotopes for diagnosis and therapy, factors to encourage recruitment of monocytes, such as cytokines, factors which inhibit the upregulation of the monocyte-ELAM, and the like.

The antibodies or other epitope-binding molecules used in the method of the present invention are preferably administered to individuals, preferably mammals, in a manner that will maximize the likelihood of the antibody or other epitope-binding molecule reaching the targeted endothelial cell, binding to it, and thereby blocking the binding of circulating monocytes. This in turn will inhibit or divert monocyte traffic through particular sites and thus control certain neoplastic or dysfunctional diseases, such as those identified in Table 1. As indicated above, carbohydrates may find use to act as inhibitors, as well as other molecules which specifically bind to the monocyte-ELAM.

The dose for individuals of different species and for different diseases is determined by measuring the effect of the antibodies or other epitope-binding molecules on the lessening of these parameters which are indicative of the disease being treated. The antibodies or other epitope- binding molecules will normally be administered parenterally, preferably intravenously. Doses of antibodies in a mouse model will generally range from about 0.5-2 mg/host/week for from about 1 to 4 weeks. The dose of the antibody or other epitope-binding molecule may have to be repeated periodically depending upon the particular disease.

When administered parenterally, the antibodies or other epitope-binding molecules will be formulated in an injectable dosage form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and Hanks' solution. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. The vehicle may contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g. buffers and preservatives. The antibody or other epitope-binding molecule is preferably formulated in purified form substantially free of aggregates and other proteins at concentrations of about 1-50 mg/ml. Suitable pharmaceutical vehicles and their formulations are described in Remington's

Pharmaceutical Sciences, by E.W. Martin, which is incorporated herein by reference.

The following examples are offered by way of illustration and not by way of limitation.

Example 1: Production of monoclonal antibodies binding to monocyte-ELAM.

bEnd3 cells (a mouse brain-derived polyoma middle T antigen transformed endothelial cell line) , provided by Werner Risau (Munich) was stimulated with LPS at 1 μg/ml. Four hours after stimulation, 5 x 10⁶ bEnd3 cells were used to immunize Fisher F344 rats. The cells were suspended in sterile, nonpyrogenic, phosphate buffered saline, and 200 μl of the suspension was injected sub-cutaneously in 4 sites (50 μl per site) . The cells were reinjected at 3 week intervals^ a total of 4 injections. The rats were sacrificed by cervical dislocation, and their spleens removed. Splenocytes were fused by traditional fusion procedures using the myeloma line SP2/0 as the fusion partner.

Hybridoma supernatants were screened for their ability to inhibit WEHI78/24 (mouse monocytoid cell line) binding to bEnd3 cells. bEnd3 cells were grown on 1cm X 1cm glass chamber slides. The cells were stimulated for 4 or 18 hours with 1 μg/ l LPS, then washed twice with PBS, and incubated with hybridoma supernatants for 30 minutes at 4°C. WEHI cells (3 X 10⁵) were added, and allowed to bind for 30 minutes on a rocking platform at 4°C. Slides were washed once with PBS to remove unbound cells, then placed in PBS with 1% gluteraldehyde to fix the remaining, bound cells. The number of WEHI 78/24 cells bound per field was enumerated by light microscopy.

Several hybridomas were found to secrete monoclonal antibodies that blocked WEHI78/24 binding to 4 and 18 hour LPS-stimulated bEnd3 cells. Three hybridomas (LM151, LM99 and LM141) recognized bands on Western blots of non-reducing SDS-PAGE gels at about 45-49 kD. The hybridomas blocked WEHI78/24 binding to stimulated bEnd3 endothelial cells at 25°, as well as 4°C. The antibodies inhibited WEHI78/24 binding, but not thymocyte binding.

The LM151 antigen was found to be expressed at low levels by unstimulated endothelium and substantially induced on activation of endothelium with IL-1, TNF-α or LPS. The surface expression of the antigen on unactivated vs. activated cells was determined by indirect immunofluorescence and flow cytometric analysis. Example 2: Monocvtoid cell interactions with cvtokine- stimulated EC.

Following activation of bEnd3 cells with LPS, IL-1 or TNF-α, as described above, a dramatic increase in binding of WEHI78/24 cells is observed: Increased binding reaches a maximum by 6 h of stimulation and remains unchanged for up to 72 h. This is determined using the assay described in Example 1, but using 2, 4, 6, 8, 12, 24, 48 or 72 hrs. of LPS stimulation. This may be contrasted with kinetics of neutrophil binding, which peak at 4-6 h and return to baseline levels at 12 h.

Blocking studies with monoclonal antibodies specific for known surface adhesion elements were performed with

WEHI78/24 cells and LPS-stimulated bEnd3 cells.

Anti-integrin subunit α4 antibodies were partially able to block WEHI78/24 binding to LPS-stimulated bEnd3 cells at

4°C. None of the other antibodies against known cell adhesion molecules, LFA-1, MAC-1 and LECAM-1 (L-selection) , as well as control antibody against T200 (the common leukocyte antigen) , are able to block and none of the antibodies (including anti-α4) are able to block binding at room temperature. Furthermore, antibodies to ICAM-1 also failed to inhibit binding at 4° and 37°C, while anti-VCAM-1 was able partially to inhibit at 4°C. In contrast, lymphocyte binding under these conditions is substantially inhibited by anti-α4 and anti-VCAM-1 monoclonal antibodies. Parallel studies with human cells were performed with human umbilical vein endothelial cells (Gimbrone et al. (1976)

"Culture of vascular endothelium" in Progress in Hemostasis and Thrombosis. Vol. 3, Ed. T.H. Spaet, New York, Grune and Stratton, pp. 1) . WEHI78/24 and the human monocyte-like line U937 were examined with LPS-stimulated HUVEC. Neither line was found to bind well to unstimulated HUVEC and binding of both cell types is significantly increased following stimulation with LPS, TNF-α and IL-1.

Anti-α4 MAb was able to block U937 binding at later time points (after about 10 hours) at 4°C, but not at 37°C. Known blocking antibodies against integrin subunit β2 and ICAM-1 failed to block binding of U937 cells to unstimulated, 4 h or 24 h LPS-stimulated HUVECs at 4° or 37°C. Monocyte binding to HUVECs stimulated with LPS for 24-48 h does not appear to involve ELAM-1, since neuraminidase treatment of U937 cells destroys the carbohydrate ligand of ELAM-1 and diminishes binding of endothelial cells (ECs) treated with LPS for 4 h, but has no effect on U937 binding after 24 or 48 h of LPS stimulation.

To determine the effect of minimally modified-low density lipoprotein (MM-LDL) on monocyte adhesion, second passage HUVECs were either untreated, treated with MM-LDL (10 μg/ml) or with MM-LDL in the presence of cycloheximide (1 μg/ml) . Human monocytes purified by elutriation were added for 30 minutes at room temperature. Nonadherent cells were washed away and adherent cells were visually counted. HUVECs treatedwith MM-LDL exhibit increased human monocyte binding reaching a maximum under 6-10 h, with the increased binding persisting for 72 h and being inhibited by the presence of cycloheximide.

Example 3: cDNA library construction/bacterial expression cloning.

Total RNA is isolated from LPS-stimulated bEnd3 cells (expressing the 151-ELAM antigen by im unofluorescence) by a single step acid guanidinium thiocyanate procedure (Chomczynski et al. (1987) Anal. Biochem. 162:156) . The RNA is further purified by overnight centrifugation over cesium trifluoroacetate (Pharmacia) . Polyadenylated RNA is purified by two rounds of selection using biotinylated oligo-dT and paramagnetic streptavidin particles (Promega) . The poly[A⁺] fraction is then used for cDNA synthesis.

The unizap XR cloning system (Stratagene) is employed. The fragments cloned into this vector are rescued with helper phage and recircularized to generate subclones into the Bluescript Sk-phagemid. Single stranded plasmid molecules are obtained by coinfection of E. coli carrying the phagemid with M13 helper phage. The single stranded DNA is rescued by retransformation into E. coli, aiding in the subsequent generation of cDNA libraries enriched for LPS-inducible transcripts by subtractive hybridization. First strand cDNA is synthesized with an XhoI-dT primer, MuLV reverse transcriptase, and methyl dCTP. After second strand synthesis, ligation of EcoRI adapters and EcoRl restriction digests, the cDNA is size selected by Sephacryl-S400 spin chromatography. The large cDNAs (greater than 500 bp) are then ligated into the unizap vector, packaged in vitro, and titered. The library comprises 10⁷ independent recombinants with a size range of 700 bp to 2 kb from the bEnd3 cell line.

The unizap cDNA library is plated out on E. coli Sure (Stratagene) at about 50,000 plaques/plate and incubated 4 h at 42°C. Duplicate nitrocellulose filters coated with

IPTG are applied and the plates are incubated for 4 h per filter at 37°C. Replica filters are washed, blocked with

BSA and probed with anti-151-ELAM antisera pre-absorbed to remove reactivity with E. coli proteins. Positive clones are identified with an alkaline phosphatase anti-rat IgG, rescreened and plaque purified. (See Goldstein et al.

(1989) Cell. 56:1063.)

Example 4: Eukarvotic expression cloning.

In this procedure, the COS cell expression/immunoselection system developed by Brian Seed is employed (Seed and Aruffo (1987) Proc. Natl. Acad. Sci. USA. 84:3365). The cDM8 vector called pcDNA-1 (Invitrogen) is employed. It is characterized by having (1) a strong promoter element composed of human cytomegalovirus immediate early enhancer sequences fused to Avian sarcoma virus long terminal repeat sequences which provides high level expression in mammalian cells; (2) a small size (4.8 kb) and an origin of replication which allows for high level replication in mammalian cells; and (3) a cloning site which contains compatible restriction sites with the cDNA utilized in the unizap system so as to directly ligate cDNA from the unizap system into the subject vector. The cDNA library is transferred into competent bacteria, followed by transfection into 50% confluent COS cells by polyethylene glycol promoted spheroplast fusion. Seventy-two hours after transfection, the cells are harvested by detaching without trypsin and selected for 151-ELAM expression by panning with monoclonal antibody coated plates and confirming with FACS. Immunoselected cells are lysed, and DNA is transformed back into E. coli for additional rounds of transfection/immunoselection.

Example 5: Production of monoclonal antibodies against stimulated human aortic endothelial cells ("HAEC") .

Mice are immunized with 24 hour LPS stimulated HAEC as described previously for the immunization of rats with LPS stimulated bEnd3 cells. Hybridoma supernatants are screened initially by immunofluorescence for reactivity with stimulated EC, but no reactivity with unstimulated EC. Unstimulated, and 24 hour LPS stimulated HUVECS were stained with hybridoma supernatant, washed to remove unbound antibody, incubated with fluorescently labelled anti-rat antibody, washed to remove unbound antibody, and their fluorescence measured by flow cytometry.

Positive supernatants are tested for their ability to block monocyte adhesion to stimulated EC employing U937 as the human monocytic cell and WEHI78/24 as the mouse monocytic cell. Positive results are confirmed using human peripheral blood monocytes.

The procedure described above is repeated with MM-LDL stimulated HAEC.

Example 6: Isolation of antigens.

Salt/detergent extracts of stimulated EC are prepared and screened with ELISA or dot blots for the detection of the antigen. The cells are initially lysed with RIPA (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate) or CHAPS (3- [cholamidopropyl-10-dimethyl ammonio]-propanesulfonate) , 0.1% SDS, 50 mM Tris, pH 8.0). Included in the lysis buffer is a protease inhibitor cocktail consisting of 0.5 mM PMSF, 1 μg/ml aprotinin, 2 mM EDTA, 1 μg/ml pepstatin, and N-ethyl maleimide. The lysate is analyzed by non-reducing and reducing SDS-PAGE and Western blot analysis.

The antigen is immunoprecipitated from extracts of unlabeled, metabolically labeled and iodinated endothelial cells in the manner as described (Berg et al. (1991) J. Cell Biol.. 114:343; Streeter et al. (1988) Nature, 331:41). Affinity columns to which are conjugated monoclonal antibodies specific for the antigen are prepared by conjugation of the antibodies to Sepharose 4B. Columns are washed in lysis buffer containing /S-octylglucoside (j8-0G) . Antigens are separated on columns using high pH (100 mM triethylamine, pH 11.5), low pH (100 mM glycine, ph 2-4), and high salt (5 M LiCl and 500 mM NaCl or KC1) . Fractions are monitored for protein content and binding to the monoclonal antibodies as well as the cell type having the complementary surface membrane protein.

Example 7: Cell adhesion assays.

Membrane adhesion molecules are eluted from affinity columns in a dialysable detergent (3-OG at 1.5-2X critical micelle concentration (CMC; 50 mM S-OG)) . The soluble glycoprotein is concentrated by Amicon filtration and 10-20 μl is added to glass wells of chamber slides (LABTEK, Wilmington, MA) containing 40-60 μl of PBS to dilute the detergent below its CMC, wherein the protein binds to the glass. After incubating for 2 h at room temperature, slides are blocked with Dulbecco's modified Eagles medium (DMEM, Applied Scientific, San Francisco, CA) containing 10 mM Hepes and 5% newborn calf serum (GIBCO Laboratories, Grand Island, NY) . WEHI78/25 cells, human peripheral blood mononuclear cells, human neutrophils, mouse peripheral lymph node (PLN) , mesenteric lymph node (LN) and Peyer's patch lymphocytes are applied to the wells. After incubation at 20 min at 4°C, room temperature or 37°C on a rocking platform, the tops of the slides are removed and slides washed by dipping twice in coplin jars of DMEM and then fixed by incubation in 1.5% glutaraldehyde in DMEM for 1 h.

Human mononuclear and polymorphonuclear cells are isolated from peripheral blood by 1G sedimentation of red blood cells with 0.6% Dextran T500 (Pharmacia, Inc.) followed by centrifugation of the leukocyte-rich supernatant on a discontinuous gradient of 42% and 51% Percoll (Haslett et al. (1985) American J. Pathol.. 119:101). Monocytes are separated from the mononuclear cell fraction by a modification of the Recalde method (Fogelman et al. (1988) J. Lipjd Res.. 29:1243) or by elutriation. Monocyte versus lymphocyte binding wells containing mixed human mononuclear cells are assessed by morphological analysis of Wright stained slides and by flow cytometric analysis of cells removed from the slide by EGTA treatment and stained with lymphocyte specific (anti-Leu4 for T cells and Dako-panB for B cells) and monocyte specific (CD14) monoclonal antibodies.

Binding of antigen to monocytes establishes the monocyte-ELAM antigen, while binding HUVEC or HAEC establishes the monocyte cell adhesion molecule.

It is evident from the above results, that a number of important diseases can be treated or diagnosed by being able to detect the presence of inflamed endothelial cells which bind to monocytes, to be able to direct specific biological active compounds to the site, and to be able to modulate the interaction between the endothelial cells and the monocytes.

In this manner, one may be able to alleviate such diseases as atherosclerosis, allergies, autoimmune diseases, certain malignancies, arthritis, inflammatory bowel diseases, transplant rejection and reperfusion injury. By using the subject compositions, by themselves or in conjunction with the modulation of other leukocyte binding events, one may be able to specifically control inflammatory episodes over extended periods of time.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described.in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for modulating the binding of monocytes to a reciprocal binding member of an endothelial cell, said method comprising: combining a medium and a compound, said medium comprising monocytes comprising a surface membrane protein capable of binding to an endothelial cell adhesion molecule, and endothelial cells capable of upregulation or activation to increase the number of cell adhesion molecules binding to monocytes, and said compound being capable of binding to said monocyte surface membrane protein or endothelial cell adhesion molecule and inhibiting binding of said monocyte to said endothelial cell.

2. A method according to Claim 1, wherein said compound is an antibody specific for said endothelial cell adhesion molecule.

3. A method according to Claim 1, wherein said compound is a fragment of said monocyte surface membrane protein and capable of binding to said endothelial cell adhesion molecule.

4. A method according to Claim 1, wherein said medium is blood.

5. A method according to Claim 1, wherein said monocytes and endothelial cells are human cells.

6. A method for modulating the binding of monocytes to a reciprocal binding member of an endothelial cell in culture, said method comprising: combining a culture medium and a compound, said medium comprising monocytes comprising a surface membrane protein capable of binding to an endothelial cell adhesion molecule, and endothelial cells capable of upregulation to increase the number of cells adhesion molecules binding to monocytes, and said compound being capable of binding to said monocyte surface membrane protein or endothelial cell adhesion molecule and inhibiting of said monocyte to said endothelial cell.

7. A method according to Claim 6, wherein said compound is an antibody specific for said endothelial cell adhesion molecule.

8. A method according to Claim 6, wherein said compound is a fragment of said monocyte surface membrane protein and capable of binding to said endothelial cell adhesion molecule.

9. A method according to Claim 6, wherein said medium is blood.

10. a method according to Claim 6, wherein said monocytes and endothelial cells are human cells.

11. A method for modulating the binding of monocytes to a reciprocal binding member of an endothelial cell, said method comprising: combining a compound with a human blood medium, said medium comprising monocytes comprising a surface membrane protein capable of binding to an endothelial cell adhesion molecule, and stimulated endothelial cells capable of upregulation to increase the number of cell adhesion molecules binding to monocytes, and said compound being capable of binding to said endothelial cell adhesion molecule and inhibit binding to said monocyte to said endothelial cell.

12. A method according to Claim 11, wherein said compound is an antibody.

13. A method according to Claim 11, wherein said antibody is a monoclonal antibody produced in immune response to bEnd3 cells.

14. A method for isolating proteins capable of binding to an endothelial cell adhesion molecule binding to monocytes, said method comprising: combining a mixture of proteins encoded by monocytes with monoclonal antibody produced in immune response to bEnd3 cells; and isolating protein which binds to said antibody.

15. A monoclonal antibody which binds to an endothelial cell adhesion molecule which binds to monocytes and blocks binding of said monocytes to endothelial cells.

16. A monoclonal antibody according to Claim 15, wherein said antibody was produced in immune response to bEnd3 cells.

17. A method for directing a compound to endothelial cells having an endothelial surface membrane protein capable of binding monocytes, said method comprising: combining a compound with said endothelial cells, said compound characterized by comprising (1) an antibody or binding fragment thereof specific for said surface membrane protein or at least the binding fragment of a monocyte surface membrane protein capable of binding to said endothelial surface membrane proteins; and (2) a compound of interest.

18. A method according to Claim 1, including the additional step of adding stimulation for upregulating said endothelial surface membrane proteins.

19. A protein composition characterized by: (1) having a molecular weight of about 45-50 kD on a Western blot with monoclonal antibodies LM99, LM141 or LM151,* (2) A surface protein of an endothelial cell, substantially absent on resting endothelial cells in vivo: and

(3) blocks binding of monocytes to activated endothelial cells and biologically active fragments thereof.

20. A cDNA encoding a protein according to Claim 19 or an active fragment thereof.

21. An expression vector comprising said cDNA according to Claim 20, under the transcriptional and translational regulation of transcriptional and translational initiation and termination sequences.

22. A second cDNA sequence having at least about 70% homology with said cDNA sequence according to Claim 20, and encoding a protein capable of blocking binding of monocytes to activated endothelial cells.