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WO1993019784A1 - Nouvelle molecule d'adherence endotheliale pour monocytes - Google Patents

Nouvelle molecule d'adherence endotheliale pour monocytes Download PDF

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Publication number
WO1993019784A1
WO1993019784A1 PCT/US1993/003153 US9303153W WO9319784A1 WO 1993019784 A1 WO1993019784 A1 WO 1993019784A1 US 9303153 W US9303153 W US 9303153W WO 9319784 A1 WO9319784 A1 WO 9319784A1
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WIPO (PCT)
Prior art keywords
binding
monocytes
cells
endothelial
monocyte
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PCT/US1993/003153
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English (en)
Inventor
Leslie M. Mcevoy
Eugene C. Butcher
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The Board Of Trustees Of The Leland Stanford Junior University
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Publication of WO1993019784A1 publication Critical patent/WO1993019784A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C07K14/70564—Selectins, e.g. CD62
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C07K16/2854—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72 against selectins, e.g. CD62
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the field of this invention is the modulation of monocyte and endothelium response to inflammation and trafficking of onocytes to sites of inflammation.
  • leukocytes An important physiological process for monitoring and treating diseased states involves the migration of leukocytes from the vascular system to the site of injury.
  • different types of cells may be recruited.
  • different groups of leukocytes such as lymphocytes, neutrophils, or monocytes, or combinations thereof, may be involved.
  • 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.
  • 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.
  • 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.
  • 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- ⁇ , minimally modified-low density lipoprotein (by minimal modification is intended storage or mild oxidation) , and the like.
  • 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.
  • nucleic acid sequences encoding the protein(s) associated with the specific binding of monocytes during this period 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.
  • a vector such as a plasmid or virus
  • 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 10 6 to 10 8 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • the antibody will be further characterized by not interfering with binding of lymphocytes and neutrophils to the endothelial cells.
  • 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- ⁇ , 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.
  • primate particularly human
  • domestic animals e.g. murine, bovine, equine, canine, feline, ovine, porcine, etc.
  • 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.
  • Poststreptococcal Diseases e.g. Cardiac manifestations of rheumatic fever
  • 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.
  • 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.
  • an appropriate salt solution or aqueous/organic gradient e.g., acetonitrile, ethanol, etc.
  • 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 ⁇ -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.
  • the cDNA sequence may be used to probe a genomic library to identify the genomic gene encoding the subject monocyte-ELAM.
  • fragments of the monocyte-ELAM may be used to bind to the monocytes to inhibit binding to the endothelial cells.
  • 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.
  • 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.
  • 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.
  • the antibodies or other epitope-binding molecules 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.
  • 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
  • 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 ⁇ g/ml. Four hours after stimulation, 5 x 10 6 bEnd3 cells were used to immunize Fisher F344 rats. The cells were suspended in sterile, nonpyrogenic, phosphate buffered saline, and 200 ⁇ l of the suspension was injected sub-cutaneously in 4 sites (50 ⁇ l 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 ⁇ g/ l LPS, then washed twice with PBS, and incubated with hybridoma supernatants for 30 minutes at 4°C.
  • WEHI cells (3 X 10 5 ) 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.
  • 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- ⁇ 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.
  • Anti-integrin subunit ⁇ 4 antibodies were partially able to block WEHI78/24 binding to LPS-stimulated bEnd3 cells at
  • Anti- ⁇ 4 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 ⁇ 2 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.
  • HUVECs minimally modified-low density lipoprotein (MM-LDL) on monocyte adhesion
  • MM-LDL minimally modified-low density lipoprotein
  • 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.
  • 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.
  • 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 10 7 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 IPTG are applied and the plates are incubated for 4 h per filter at 37°C. Replica filters are washed, blocked with
  • Example 4 Eukarvotic expression cloning.
  • 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.
  • 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.
  • 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 ⁇ g/ml aprotinin, 2 mM EDTA, 1 ⁇ g/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 ⁇ l is added to glass wells of chamber slides (LABTEK, Wilmington, MA) containing 40-60 ⁇ l 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) .
  • DMEM Dulbecco's modified Eagles medium
  • 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.
  • diseases as atherosclerosis, allergies, autoimmune diseases, certain malignancies, arthritis, inflammatory bowel diseases, transplant rejection and reperfusion injury.
  • 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.

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Abstract

L'invention concerne des procédés et des compositions qui permettent de moduler la fixation de monocytes à des cellules endothéliales, en particulier au cours d'épisodes inflammatoires. Lesdites compositions se fixent à la protéine membranaire superficielle du moncyte et/ou à la protéine membranaire superficielle endothéliale qui sont complémentaires ou entraînent l'adhérence du monocyte à la cellule endothéliale. Ces compositions peuvent être utilisées à des fins diagnostiques ou thérapeutiques.
PCT/US1993/003153 1992-04-07 1993-04-06 Nouvelle molecule d'adherence endotheliale pour monocytes WO1993019784A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997000956A1 (fr) * 1995-06-20 1997-01-09 Trustees Of Boston University Molecules d'adherence reagissant a l'hypoxemie, anticorps specifiques et leurs utilisations
US7594535B2 (en) * 2005-07-25 2009-09-29 Castrip, Llc Twin roll caster, and equipment and method for operating the same

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J. CLIN. INVEST., Volume 76, issued November 1985, M.P. BEVILACQUA et al., "Interleukin 1 Acts on Cultured Human Vascular Endothelium to Increase the Adhesion of Polymorphonuclear Leukocytes, Monocytes, and Related Leukocyte Cell Lines", pages 2003-2011. *
J. CLIN. INVEST., Volume 85, issued April 1990, J.A. BERLINER et al., "Minimally Modified Low Density Lipoprotein Stimulates Monocytes Endothelial Interactions", pages 1260-1266. *
TRANSPLANTATION, Volume 48, issued November 1989, M.A. JUTILA et al., "Inflammation-Induced Endothelial Cell Adhesion to Lymphocytes, Neutrophils, and Monocytes", pages 727-731. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997000956A1 (fr) * 1995-06-20 1997-01-09 Trustees Of Boston University Molecules d'adherence reagissant a l'hypoxemie, anticorps specifiques et leurs utilisations
US7594535B2 (en) * 2005-07-25 2009-09-29 Castrip, Llc Twin roll caster, and equipment and method for operating the same

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