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EP0675735A1 - Procede de modulation de la migration transendotheliale des cellules favorisant l'inflammation et procedes connexes de mesure de cette migration - Google Patents

Procede de modulation de la migration transendotheliale des cellules favorisant l'inflammation et procedes connexes de mesure de cette migration

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Publication number
EP0675735A1
EP0675735A1 EP94907806A EP94907806A EP0675735A1 EP 0675735 A1 EP0675735 A1 EP 0675735A1 EP 94907806 A EP94907806 A EP 94907806A EP 94907806 A EP94907806 A EP 94907806A EP 0675735 A1 EP0675735 A1 EP 0675735A1
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EP
European Patent Office
Prior art keywords
pecam
leukocytes
inflammation
transendothelial migration
agent
Prior art date
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EP94907806A
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German (de)
English (en)
Inventor
William A. Apartment 14D Muller
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Rockefeller University
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Rockefeller University
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Publication of EP0675735A1 publication Critical patent/EP0675735A1/fr
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    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to the migration of leukocytes and like cells across the endothelium, and more particularly, to the modulation of such transmigration and its effect on inflammation.
  • Inflammation is the response of vascularized tissues to infection or injury. Clinically it is accompanied by four classic signs: redness, heat, pain and swelling. Its course may be acute or chronic.
  • inflammation involves the adhesion of leukocytes (white blood cells) to the endothelial wall of blood vessels and their infiltration into the surrounding tissues (Harlan, 1985).
  • Acute inflammation is characterized by the adhesion and infiltration of polymorphonuclear leukocytes (PMN) (Harlan, 1987 and Malech and Gallin, 1987). PMN accumulation in the tissues begins between two and one half to four hours after an inflammatory stimulus and reaches its peak or ceases at about one to two days (Bevilacqua and Gimbrone, 1987).
  • Chronic inflammation is characterized by the adhesion and infiltration of other leukocytes, especially monocytes and lymphocytes.
  • Leukocytes leave the circulation by binding to the luminal surface of the vascular endothelium then migrating between tightly apposed endothelial cells into the body tissues (Marchesi, 1961; Marchesi and Florey, 1960). This process is constitutive in some situations. For example, monocytes exit the circulation at a low frequency to become tissue macrophages (van Furth, 1986). Circulating lymphocytes enter lymph nodes by binding to and migrating across the specialized high endothelial venules (HEV) in lymphoid tissue (Lasky, 1992; Stoolman, 1989).
  • HEV high endothelial venules
  • This process involves recognition and binding of L-selectin to specialized, lymph node restricted counter-receptors on the HEV (Gallatin et al., 1983; Gallatin et al., 1986).
  • leukocytes emigrate in large numbers into the inflammatory site, by recognizing and binding to cytokine induced cell adhesion molecules (CAMs) on the endothelium (Springer, 1990; Osborn, 1990; Pober and Cotran, 1990).
  • CAMs cytokine induced cell adhesion molecules
  • a related process may occur during early atherogenesis when monocytes selectively emigrate into the subintima of affected arteries (Ross, 1986).
  • infiltrating leukocytes In normal inflammation, the infiltrating leukocytes phagocytize invading organisms or dead cells, and play a role in tissue repair and the immune response. However, in pathologic inflammation, infiltrating leukocytes can cause serious and sometimes lethal damage.
  • Rheumatoid arthritis and atherosclerosis are examples of chronic inflammatory diseases in which mononuclear leukocytes infiltrate the tissues and cause damage (Hough and Sokoloff, 1985 and Ross, 1986).
  • Multiple organ failure syndrome, adult respiratory distress syndrome (ARDS), and ischemic reperfusion injury are acute inflammations in which infiltrating PMNs cause the damage (Harlan, 1987 and Malech and Gallin, 1987).
  • ARDS Adult respiratory distress syndrome
  • pneumococcal meningitis mortality can be directly correlated with the amount of meningeal inflammation (McAllister et al., J. Infect. Dis. , 132:355- 360, 1975).
  • McAllister et al., J. Infect. Dis. , 132:355- 360, 1975 a method of dampening inflammation during the course of therapy with an antibiotic would be advantageous in treating infections, particularly endotoxic shock and ARDS associated with infection.
  • inflammatory agents can act on the leukocytes, making them hyperadhesive for endothelium.
  • Known inflammatory agents include leukotriene-B4 (LTB4), complement factor 5a (C5a), and formyl-methionyl-leucyl-phenylalanine (FMLP).
  • LLB4 leukotriene-B4
  • C5a complement factor 5a
  • FMLP formyl-methionyl-leucyl-phenylalanine
  • J ⁇ integrins proteins now known as J ⁇ integrins.
  • the ⁇ 2 integrins are dimers of the CD 11 and CD18 proteins.
  • CDlla/CD18 also called LFAl
  • ICAM1 intercellular adhesion moleculel
  • inflammatory agents act directly on endothelial cells to substantially augment leukocyte adhesion.
  • these agents include the cytokines interleukin-1 (IL-1), and tumor necrosis factor (TNF), as well as the bacterial endotoxin, lipopolysaccharide (LPS).
  • IL-1 has been shown to stimulate adhesion of PMN, monocytes, and the related cell lines HL-60 (PMN-like) and U937 (monocyte-like), to human endothelial cell monolayers.
  • the action is both time-dependent and protein-synthesis dependent (Bevilacqua et al. , 1987a; Bevilacqua et al., 1987b; and Bevilacqua et al., 1985).
  • cytokines may have one of several effects on endothelial cells vis a vis surface adhesion molecules. They may induce de novo synthesis of adhesion molecules, such as in the case of IL-1 and TNF, inducing expression of E-selectin and VCAMl (Osborn, 1990). They may also augment expression of pre-existing adhesion molecules such as the effect of these two cytokines on ICAM1. They may cause redistribution of pre-existing adhesion molecules from an intracellular compartment to the cell surface by degranulation, as in the case for the surface expression of P-selectin after stimulation of endothelial cells by thrombin or histamine.
  • PECAM platelet/endothelial cell adhesion molecule- 1 or PECAM- 1
  • PECAM- 1 PECAM-1
  • the sequence of PECAM is identified and recombinant human PECAM-1 (rhPECAM-1) is disclosed.
  • the primary thrust of the disclosure, including reference to antibodies to PECAM- 1, relates to the activity of the discovered molecule as it relates to the potential binding of platelets to endothelial cell membranes.
  • the use and activity of PECAM was disclosed from the perspective of platelet surface recognition and binding.
  • the present inventor has studied cellular migration across the intercellular zone of the endothelium, and in Muller et al. (1989), J. Exp. Med. , 170:399-414, generated a murine monoclonal antibody against human endothelial cell membrane proteins that were present in greater concentrations at the junction between endothelial cells. At that time, however, there was no indication as to the exact role played by the plasmalemmal proteins that were identified, nor could there be any sense of the role that an antibody thereto might play.
  • the findings of Schimmenti et al. that PECAM- 1 inhibited the rate of sheet migration were cumulative with those of Albelda et al. and did not extend beyond the interactions between endothelial cells.
  • the studies of Muller et al. focused primarily on monocyte migration and concluded that the adhesion involving the ⁇ 2 integrins is the primary event that must precede monocyte TEM. As such, the authors concluded that the
  • Bogen et al. in fact, offer evidence that teaches away from the present invention. For example, they provide immunoelectron microscopic data (See Bogen et al. , Figures 4 and 5) that are interpreted as demonstrating that lymphocytes express less PECAM as they come closer to migrating across endothelial cell boundaries. This is consistent with the attribution to PECAM of a negative regulatory role in transmigration, as it suggests that lymphocytes have to rid themselves of PECAM before transmigration can take place.
  • mice were found to have a deficiency in rolling (Mayadas et al., 1993). Furthermore, anti-selectin reagents were demonstrated to block rolling in several other in vivo models (Ley et al., 1991; von Andrian et al. 1991). Antibodies against the leukocyte integrin CD11/CD18 complex were demonstrated to block tight apical adhesion of human leukocytes to human endothelial cells (Harlan, et al. 1985) in vitro. The same or similar antibodies also prevented this adhesion in vivo (Arfors et al. 1987).
  • the present invention concerns a method and associated agents and compositions for modulating the transmigration of cells such as leukocytes into sites of acute and/or chronic inflammation during infectious or non-infectious conditions, the method comprising administering a therapeutic amount of an agent or composition selected from the group consisting of antibodies to PECAM, antagonists of PECAM activity, recombinant human PECAM-1 (rhPECAM-1), and active fragments thereof.
  • an agent or composition selected from the group consisting of antibodies to PECAM, antagonists of PECAM activity, recombinant human PECAM-1 (rhPECAM-1), and active fragments thereof.
  • PECAM activity antagonists may comprise materials such as molecules, including small organic or inorganic molecules, polypeptides and fragments of same, that are structurally unrelated to PECAM or anti-PECAM antibodies, but which work through, or have as a basis of their therapeutic action the ability to interfere with the function of PECAM as defined herein. Such antagonists could interact with PECAM wherever found, and may accordingly interact with the leukocyte or the endothelial cell.
  • a specific anti-PECAM antibody comprises the monoclonal antibody hec7 and particularly the antibody prepared from Hybridoma hec7.2 deposited with the American Type Culture Collection on December 23, 1992 and assigned ATCC Accession No. HB 11227, and/or active fragments thereof.
  • Hybridoma hec7.2 is one of four identical subclones, hec7.1-hec7.4, that have been prepared by the inventor, and the invention is accordingly intended to extend in its scope to the use of any one of these subclones.
  • this invention concerns a method and associated materials for inhibiting the ingress of leukocytes into tissues in patients suffering from either acute or chronic conditions which are associated with undesirable inflammation, by administration of a therapeutic amount of one of the agents of the present invention to a patient in need of such therapy.
  • the preferred antibodies are as set forth above.
  • This invention also concerns a method and associated materials for eliminating or reducing inflammation in a patient wherein the patient is being administered an anti-infective agent for an infectious disease, which comprises the administration prior to, along with or after the anti-infective agent of a therapeutic amount of one of the present agents. Dosage forms combining an anti-infective agent and a therapeutic amount of the present agent are also described.
  • the present invention also extends to a method and associated assays for measuring and monitoring transendothelial migration for the purposes of diagnosing an alteration in PECAM function, the method comprising disposing a quantity of a cellular agonist such as leukocytes, serum or plasma taken from a patient under such examination, along a monolayer of human endothelial cells grown from cultured HEC. The cells are maintained in contact with the monolayer for approximately 1 hour, which should be sufficient for normal transmigration to take place.
  • a cellular agonist such as leukocytes, serum or plasma taken from a patient under such examination
  • the monolayers are then fixed, stained with AgNO 3 and Wright Giemsa stain, and thereafter subjected to Normarski or Hoffman optics to spatially visualize the exact location of cells disposed within the intercellular EC junctions, after which the results are compared to a predetermined continuum of such measurements developed from subjects ranging in condition from normal to the presence of the condition or conditions under diagnosis or observation.
  • the manner in which the cells are labeled may vary and includes radiolabeling of the leukocytes or by the use of colorimetric tracers.
  • the method may also be automated as disclosed in greater detail herein.
  • the invention also contemplates a receptor assay within its scope, where all manner of antigens or other agents recognized by the PECAM receptor of the HEC may be identified and their activity measured.
  • the invention further includes a method for detecting idiopathic or known stimuli on the basis of their ability to modulate PECAM function or expression.
  • stimuli could be identified and detected by their ability to either stimulate or suppress TEM by leukocytes such as monocytes and polymorphonuclear cells.
  • leukocytes such as monocytes and polymorphonuclear cells.
  • samples of endothelial cells could be treated with/exposed to a number of known modifying agents such as anti-PECAM reagents, cytokines or the like, as a control, while parallel cellular samples could be treated with or exposed to an extract of material believed to contain such a stimulus.
  • the respective samples could then be incubated, and thereafter, control leukocytes applied to the HEC monolayers and assayed for the presence and/or extent of TEM that occurs.
  • the assay of the invention provides a precise quantitation of leukocyte transmigration that can equally precisely predict the efficacy if any, of a drug being evaluated as a potential modulator of inflammation or of the related pathological state. Accordingly, the invention extends to methods and associated assays and kits for diagnosing the likelihood of the development of alterations in PECAM function or expression, or the conditions that may cause them, or result from them, and for testing potential therapeutic agents such as drugs that may be useful as modulators of such inflammation or such conditions, and particularly, inflammation and conditions causing same wherein the deleterious condition or pathology results from the transendothelial migration of leukocytes, such as monocytes, neutrophils, lymphocytes, eosinophils and/or basophils.
  • leukocytes such as monocytes, neutrophils, lymphocytes, eosinophils and/or basophils.
  • Another object of the present invention is to afford a method of treating a patient afflicted with a condition presenting concomitant acute inflammation, such as endotoxic shock or adult respiratory distress syndrome, by administering a therapeutic amount of an anti-PECAM antibody or soluble recombinant PECAM, or active fragments thereof, whereby the amount of the influx of leukocytes into the affected tissue is eliminated or greatly reduced.
  • a condition presenting concomitant acute inflammation such as endotoxic shock or adult respiratory distress syndrome
  • FIGURE 1 is a composite of photomicrographs showing that anti-PECAM antibody blocks transendothelial passage of monocytes.
  • Peripheral blood mononuclear cells PBMC
  • hec7 mAb a
  • isotype-matched control mAb b,c
  • FIGURE 2 is a composite of scanning electron micrographs depicting that hec7 anti-PECAM mAb blocks migration of monocytes through the intercellular junction.
  • PBMC peripheral blood monolayers
  • Control monolayers (a) were virtually devoid of apical monocytes
  • monolayers exposed to hec7 treated PBMC were carpeted with Mo (b), most of which appeared to be bound over the intercellular junctions.
  • Mo molycerin
  • FIGURE 3 shows that anti-PECAM mAb hec7 significantly blocks transendothelial migration of Mo, whether pre-bound or added at the start of co-culture.
  • PBMC peripheral blood monolayers
  • anti-PECAM anti-PECAM
  • anti-CD 14 anti-CD 14
  • Incubation proceeded for 1 hr. at which time cells were processed as in Experimental Procedures, and the percentage of the cells remaining associated with the monolayer that had transmigrated was assessed.
  • the data displayed is the mean ⁇ standard deviation of six replicates for each experimental sample.
  • FIGURE 4 shows that hec7 is as effective as polyclonal anti-PECAM at blocking TEM.
  • PBMC peripheral blood monolayers
  • hec7 or anti-CD14 or a 1:100 dilution of rabbit anti-PECAM serum or preimmune serum.
  • Transmigration proceeded for 1 hour, and cells were processed as in Figure 3.
  • FIGURE 5 shows that hec7 Fab as well as IgG significantly blocks TEM at low concentrations.
  • Equal numbers of PBMC were added to HEC monolayers and the transmigration assay was carried out for 1 hr at 37°C in medium M199 (M199) or M199 supplemented with either anti-CD14 mAb 3C10 at 30 ⁇ g/ml (3C10) or hec7 IgG or Fab at the indicated concentrations.
  • FIGURE 6 shows that the blockage of transmigration is specific for anti-PECAM mAb.
  • Other mAb that bind to Mo surface antigens do not block TEM.
  • Equal numbers of PBMC were added to HEC monolayers for 1 hr. in M199 containing the indicated mAb at 20 ⁇ g/ml. Only hec7 significantly decreases transmigration of Mo.
  • FIGURES 7A and 7B show that hec7 does not decrease chemotaxis of Mo or PMN.
  • PBMC or PMN were isolated and resuspended in M199 + 0.1 % HSA with or without hec7 IgG or Fab at 10 ⁇ g/ml (a) or hec7 Fab at the indicated concentrations (b).
  • Leukocytes were then added to collagen gels impregnated with a) M199 or formylnorleucyl-leucyl-phenylalanine (fNLLP), or b) normal culture medium or infranate from confluent HEC cultures. After incubation for 1 hr. at 37°C, chemotaxis was quantitated as described in Experimental Procedures.
  • FIGURE 8 is a graph illustrating that inhibition of TEM by hec7 is long-lasting and reversible.
  • PBMC peripheral blood mononuclear cells
  • control mAb closed circles
  • Transmigration was assessed in the usual manner in six replicate cultures for each sample.
  • FIGURE 9 shows the results of the control experiments wherein hec7 and heel Fab fragments remain concentrated in the intercellular junctions.
  • Normal culture medium supplemented with Fab fragments of hec7 (anti-PECAM) or heel at 20 ⁇ g/ml was added to subconfluent HEC and replaced with successive feedings as the cells came to confluence over one week.
  • FIGURE 10 shows that transmigration can be blocked by anti-PECAM treatment of either Mo or HEC junctions.
  • HEC monolayers were cultured under standard conditions (Control) or in the presence of hec7 or heel Fab as described in the legend to Figure 9.
  • PBMC were resuspended in M199 containing control mAb 3C10 (open bars) or hec7 (black bars) both at 20 ⁇ g/ml, or recombinant soluble PECAM (sPECAM, hatched bars) at 1 or 10 ⁇ g/ml, and transmigration proceeded for 1 hr.
  • hec7 blocked TEM equivalently whether it was added to the Mo or the HEC, and there was no additional blockade when the two arms were combined (hec7 Fab/hec7). Soluble recombinant PECAM blocked as well as hec7, and again, was not additive with hec7 Fab bound to the HEC (hec7 Fab/sPECAM).
  • FIGURE 11 depicts the expression and isolation of recombinant soluble PECAM.
  • COS-1 cells transfected with the soluble PECAM plasmid (T) or control plasmid (C) or HEC (E) were metabolically labeled, and culture supernates (s) or cell lysates (1) were subjected to immunoprecipitation with hec7, as described in
  • the fluorogram of the SDS-PAGE gel shows authentic PECAM present in the HEC lysates, but not the supernate.
  • Transfected COS cells secrete a heavily-labeled protein of Mr « 90 kD, as expected; a smaller precursor band is detected in the detergent lysates of these cells. No protein is recognized in the control COS samples.
  • FIGURE 12 shows that hec7 blocks transmigration of Mo across cytokine activated HEC.
  • HEC were activated by culturing in the presence of 10 ng/ml TNF ⁇ for 3 hours to induce VCAM-1 and upregulate ICAM-1.
  • PBMC were added in the presence of hec7 or 3C10 mAb at 20 ⁇ g/ml, and transmigration was allowed to proceed for 1 hour.
  • FIGURE 13 shows that anti-PECAM reagents block transmigration of neutrophils.
  • HEC monolayers were cultured in the presence of 10 ng/ml TNF ⁇ for 2 hours to induce the expression of L-selectin as well as VCAM-1 and to enhance ICAM-1 expression (Muller and Weigl, 1992).
  • PMN were resuspended in warm M199 supplemented with 3C10 or hec7 at 20 ⁇ g/ml, preimmune serum or rabbit anti- PECAM serum at 1: 100, or recombinant soluble PECAM at 10 ⁇ g/ml, and added to the activated HEC monolayers for 1 hour.
  • FIGURE 14 is a series of graphs demonstrating that anti-PECAM mAb blocks leukocyte emigration.
  • Mice were injected via lateral tail vein with the indicated dose of mAb, normal hamster IgG (NHIgG), or Dulbecco's PBS (DPBS) in 0.1 ml of DPBS.
  • mAb normal hamster IgG
  • DPBS Dulbecco's PBS
  • Peritoneal cells were harvested 20 hours after the thioglycoUate challenge.
  • 2H8 - hamster anti-mouse PECAM CD31
  • 5C6 - rat anti-mouse Mac-1 CDllb
  • the graphs depict the mean and standard error of the mean for each group. (Three mice per group.) Data are expressed as the total numbers of recovered leukocytes or PMN, or as the concentrations of these cells in the peritoneal lavage fluid.
  • FIGURES 15 A and 15B each present two graphs showing that anti-PECAM- 1 mAb blocks leukocyte emigration in response to thioglycoUate injection.
  • Peritoneal cells were harvested at 24 h from mice that had received 250 ⁇ g of the indicated mAb, normal hamster IgG (N. Ham. IgG), or DPBS (None), i.v., at time zero and thioglycoUate or DPBS i.p. at 4 h.
  • Two experiments, representative of seven, are shown.
  • AKR/J mice were used in experiment 1 presented in Figure 15A, while CD2F J mice were used in experiment 2 ( Figure 15B).
  • Anti-PECAM-1 and anti-CD lib mAb block influx of PMN and mononuclear cells. Bars indicate mean ⁇ s.e.m. for each group (five mice/group).
  • FIGURES 16A-16E demonstrate that mesenteric venules from anti-PECAM-1 treated mice show increased numbers of intraluminal leukocytes in apparent contact with the endothelium.
  • Mice treated with anti-PECAM- 1 mAb as in Fig. 15 revealed a high proportion of leukocytes (predominantly PMN, arrowheads) in random sections in contact with the endothelium ( Figure 16A), while mice treated with the nonblocking anti-CD18 mAb showed rare leukocytes on the endothelial surface ( Figure 16B), despite the fact that many had transmigrated.
  • FIGURES 17A and 17B illustrate the relative paucity of mononuclear cells in the subcapsular sinus of the draining mesenteric nodes in anti-PECAM-1 treated mice.
  • Mesenteric lymph nodes from thioglycollate-treated mice were fixed and sectioned as described in Methods.
  • Figure 17A shows the subcapsular sinus (asterisk, vertical lines define width of sinus) of a representative node from mice treated with Normal Hamster IgG. Note the large numbers of mononuclear cells within the sinus, indicating their recent arrival via afferent lymphatics.
  • Figure 17B shows relatively few mononuclear cells in the same area from a mouse treated with 2H8 anti-PECAM- 1, correlating with the decreased total peritoneal cells recovered from peritoneal lavage. Both panels x 80.
  • an “agonist” is a chemical agent, compound, antigen or like material that mimics or promotes certain activity of a cellular colony, antigen, antibody, or other moiety
  • an “antagonist” is an agent, compound, or like material that counteracts, inhibits or blocks such activity.
  • the term "antagonist” specifically extends herein to those materials including compounds, agents other than antibodies to PECAM, molecules including small molecules, polypeptides, fragments of these, and the like, that are structurally unrelated to PECAM, that inhibit PECAM activity, and by so doing, exhibit therapeutic activity as is proposed for the agents of the present invention.
  • antagonists include, but are not limited to, fusion proteins wherein PECAM- 1 or portions thereof are covalently connected tin series to portions of immunoglobulins, complement regulatory proteins, or other molecules in order to confer on the PECAM antagonists additional biological properties such as longer biological halflife, increased affinity for their ligands, or the ability to be targeted to certain locations in the body.
  • antagonists also contemplates alternatively glycosylated forms of PECAM- 1, which differ on the basis of posttranslational modification of the core protein and may be so modified naturally or through appropriate biochemical manipulation in vitro. Such alternative glycoforms may differ in biological activity, biological halflife, tissue distribution, and function. Also contemplated as such antagonists are compounds of the glycosaminoglycan class and carbohydrates having similar structure.
  • PECAM- 1 is known to have a glycosaminoglycan binding site in the second immunoglobulin loop. Furthermore, this site is at or close to the site on PECAM- 1 believed to be responsible for the function of PECAM-1 in transendothelial migration.
  • glycosaminoglycans including as representative examples, but not limited to heparin and heparan sulfate, would bind to PECAM- 1 in this region and serve as antagonists of biological activity.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses, inter alia, polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may also be engineered as a molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • “Fragments of monoclonal antibodies” means the conventional Fab and F(ab') 2 fragments, as well as any chimeric or synthetic forms of antibody recognizing and binding to PECAM- 1, including humanized antibodies and fragments thereof.
  • Fab and F(ab') 2 fragments are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al.
  • Fab' antibody molecule portions are also well-known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • “Fragments of PECAM- 1” means any portions of the molecule having the same amino acid sequence as intact PECAM-1 or being comprised of such portions. This would include, but is not limited to, soluble recombinant forms of PECAM- 1 such as the one described herein, polypeptides corresponding to isolated immunoglobulin domains of PECAM- 1 or parts thereof, and polypeptides corresponding to immunoglobulin domains of PECAM- 1 or parts thereof combined with other such peptides derived from the sequence of PECAM- 1 linked in a linear or three dimensional manner that is different from their relationship in intact PECAM- 1. Also contemplated as "Fragments of PEC AM- 1" are any of the alternative glycoforms (as defined above) of the molecules aforementioned.
  • active fragments refers to fragments as generally defined herein that exhibit activity that is the same as the antibodies/antagonists to PECAM, or in the converse, exhibit the same activity as those promoters of PECAM activity, both as contemplated by the present invention. "Active fragments” likewise contemplates and includes within its scope soluble fragments of PECAM, soluble fragments of any antibodies thereto, or soluble fragments of antagonists thereof where appropriate.
  • Leukocytes wherever appearing herein including the Specification and Claims, refers to any and all classes of circulating white blood cells, including neutrophils (polymorphonuclear leukocytes (PMN)), monocytes, lymphocytes, eosinophils, and basophils, as well as malignant variants thereof that might occur in leukemia and lymphoma, in the absence of specific reference to cells of a particular class.
  • neutrophils polymorphonuclear leukocytes (PMN)
  • monocytes neutrophils
  • lymphocytes lymphocytes
  • eosinophils eosinophils
  • basophils hepatocytes
  • Inflammation refers to the response of viable vascularized tissue to injury and in particular to the influx of circulating leukocytes into such tissues, either in an acute or chronic manner.
  • inflammation include, but are not limited to, foci of infection, autoimmune injury such as a rheumatic joint or diabetic Islet of Langerhans, myocardial infarction, graft rejection, atherosclerosis, wound healing, granulomatous reactions and idiopathic inflammatory conditions.
  • PECAM will be used interchangeably with “PECAM- 1”.
  • Transendothelial migration will be used interchangeably with “transmigration” and the abbreviation “TEM” to refer to the process whereby leukocytes move across the endothelial cell lining of blood vessels by squeezing between the apposed endothelial cells. This is a well-accepted stage in the process of inflammation.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to cause by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the movement of leukocytes to a site of inflammation.
  • the present invention derives from investigations with a previously described human endothelial culture system to begin to dissect the process of leukocyte transmigration at the molecular level, as part of the further elucidation of the mechanism of inflammation.
  • selectins and integrins are known to contribute to the binding of leukocytes to the endothelium.
  • a molecule known as platelet endothelial cell adhesion molecule or PECAM- 1 has been found to be crucial to the process of transmigration through intercellular junctions, and consequently, represents a significant focus at which to intervene therapeutically in inflammation, as well as other events characterized by leukocyte movement.
  • an aspect of the invention relates to the discovery of an agent comprising a material selected from the group consisting of antibodies to PECAM, including a monoclonal antibody to PECAM, recombinant soluble PECAM, antagonists of PECAM activity, and active fragments of any of these, that may be administered to modulate or control the transendothelial migration of cellular agonists of inflammation such as monocytes or PMN.
  • the anti-PECAM antibody for example, blocks leukocyte migration by from about 70 to about 90%.
  • the antibody blocks if it is used to pretreat the leukocytes or the endothelial junctions, suggesting that PECAM mediates homophilic adhesion between leukocytes and endothelial cells.
  • anti-PECAM If endothelium is activated with cytokines, anti-PECAM still blocks migration of both monocytes and neutrophils. Anti-PECAM does not block chemotaxis of either cell type in standard assays. As discussed in greater detail later on herein, light and electron microscopy reveal that leukocytes inhibited in transmigration remain tightly bound to the apical surface of the endothelial cell and precisely over the intercellular junction.
  • This invention relates in a first aspect to inhibiting the influx of cellular agonists of inflammation such as leukocytes, into tissues and organs as a result of infectious or non-infectious conditions by the administration of a therapeutic amount of an agent capable of modulating transendothelial migration of such cellular agonists, to a patient in need of such therapy.
  • Cellular agonists contemplated by the present invention include monocytes (Mo), neutrophils (PMN), certain subtypes of T lymphocytes and a larger fraction of activated T lymphocytes, as well as certain malignant myeloid and lymphoid cell lines, all of which may express PECAM- 1.
  • inflammation may result from any of a variety of infective agents, including gram-positive and gram-negative bacteria as well as viruses, parasites and fungi, or may arise from a non-infectious source such as trauma.
  • infective agents including gram-positive and gram-negative bacteria as well as viruses, parasites and fungi, or may arise from a non-infectious source such as trauma.
  • included infections are those which are susceptible to treatment with beta-lactam antibiotics, such as influenza, meningitis, pneumococcal infections, streptococcal infections, salmonella; certain bacillus, and the like.
  • Particular infectious agents comprise Haemophilus influenzae B; N.
  • the inflamed tissues or organs which are the target of the present invention can likewise be any body tissue or organ susceptible to inflammation by the above- described agents.
  • the present invention is accordingly adaptable to the treatment, for example, of the lung, central nervous system, kidney, joints, endocardium, eyes and ears.
  • the present invention is also applicable to inflammation arising from causes other than infections, such as from artificial implants or allografts, ARDS precipitated by burns, surgery, toxic chemicals, fracture of bones, or other trauma.
  • a potential problem with the chronic use of a monoclonal antibody such as hec7 may exist. This effect may be ameliorated or obviated by using active fragments of the monoclonal antibody so as to minimize the amount of foreign protein injected.
  • Another alternative is to prepare a chimeric antibody in which the binding region of the antibody hec7 is combined with the constant regions of human immunoglobulin, or using soluble rhPECAM or active fragments thereof.
  • a further aspect of the present invention is that of reducing or eliminating the influx of leukocytes in iatrogenically induced inflammation such as may occur with the administration of an anti-infective agent.
  • the method comprises the administration prior to, along with, or after the anti-infective agent of a therapeutic amount of one of the agents of the present invention to a patient in need of such therapy.
  • a preferred agent is soluble recombinant human PECAM-1.
  • a potential therapy would comprise administration of an effective dose of anti-PECAM reagent to a patient dependent on this class of anti-inflammatory compounds.
  • anti-infective agents and particularly beta- lactam antibiotics, cause additional inflammation as a result of their therapeutic effect.
  • anti-infective agents sterilize a given infection, they cause release of toxic products, for example, the cell wall and/or endotoxin of the infecting agent.
  • Such bacterial components initiate an inflammatory response, often most acute in the lung. It is this inflammation which contributes significantly to the lung damage that is the long-term consequence of many infections.
  • agents of the present invention possess the ability to block movement of leukocytes, they are uniquely suited to treat both non-infectious causes and infectious causes.
  • Representative non-limiting instances or conditions where chronic inflammation may occur due to either infectious or non-infectious causes, and consequently, where the administration of the present agents may be beneficial include the following: the sequelae of organ transplantation or tissue allograft; atherosclerosis (arteriosclerosis); diseases known or hypothesized to be autoimmune in nature, including, but not limited to: multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Sjogren's Syndrome, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, Type I diabetes mellitus, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease including Crohn's Disease (regional enteritis) and ulcerative colitis,
  • Corresponding conditions presenting instances of acute inflammation are also capable of treatment by the therapeutic methods of the present invention.
  • the following non-limiting examples are included: pneumonia or other respiratory tract inflammation due to any cause; Adult Respiratory Distress Syndrome (ARDS) from any etiology; encephalitis, with inflammatory edema; immediate hypersensitivity reactions including, but not limited to, asthma, hayfever, cutaneous allergies, acute anaphylaxis; arteritides including, but not limited to temporal arteritis and polyarteritis nodosa; diseases involving acute deposition of immune complexes, including, but not limited to, rheumatic fever, post-infectious (e.g., post-Streptococcal) glomerulonephritis, acute exacerbations of Systemic Lupus Erythematosus; pyelonephritis; cellulitis; cystitis; acute cholecystitis, conditions producing transient ischemia anywhere along the gastrointestinal tract, bladder
  • the agents can be combined in a single unit dosage form with the anti-infective agent for convenience of administration.
  • dosage form is most preferably an intravenous dosage form since most anti-infective agents, particularly the beta-lactam antibiotics, are available in a suitable chemical form for parenteral administration, such as via the intravenous route. This is also a viable route of administration for the present agents.
  • the anti-infective agent and the agent can be combined in a single ampoule solution. Where this is not possible, the anti-infective agent and the agent can be packaged separately and mixed just prior to injection.
  • Administration can likewise be via a mixture with any standard intravenous solution, i.e., normal saline.
  • the present agents may be self-administered on a regulated basis as a form of chronic therapy, and preferably via local or oral administration.
  • the amount of anti-infective agent in the dosage form is dependent upon the particular anti-infective agent being utilized and the particular infection being treated.
  • the amount of the present agent utilized in a dosage form can range from about 1 to about 1,000 mg, with 10-100 mg per dosage unit being highly preferred. Dosages can be administered one to four times daily, with continued therapy for as long as the infection persists.
  • the method of administering the dosage unit may, of course, be varied by the treating physician due to patient condition and the severity of the infectious disease being treated.
  • An additional therapeutic protocol in accordance with the present invention is predicated on the activation and mobilization of leukocytes, monocytes and lymphocytes to treat infection or a like deleterious condition, in instances where the host exhibits immune incapacity or dysfunction related to leukocyte inaction. Since the invention demonstrates that PECAM is necessary for transendothelial migration, agents that augment or enhance PECAM function or activity may be able to ameliorate these conditions. Accordingly, appropriate compositions containing PECAM or like promoters of transendothelial migration, or agents or drugs that augment or enhance PECAM expression, function, or activity could be administered to the patient or host in need of such treatment. Promoters of PECAM would include leukocytes that have functional PECAM expressed or otherwise disposed on their surfaces in greater amounts.
  • PECAM protein astolic advantт
  • Delivery of additional PECAM could be by promotion of increased expression via increasing translation or transcription, by delivery of PECAM to the leukocyte surface via liposome encapsulation, or by like alternative measures all contemplated within the scope of the invention.
  • the method of administration would include those known procedures, including parenteral techniques as are conventionally used by skilled medical personnel. Dosages and protocol of administration would likewise vary.
  • chronic inflammatory conditions (especially in an immunocompromised host), such as tuberculosis; any acute and/or chronic inflammatory process in a person suffering from AIDS or immunocompromised due to chemotherapy, radiotherapy to the bone marrow, leukemia, or congenital immune system dysfunction; wound healing in skin, especially of stasis ulcers in a diabetic or person suffering from limb ischemia, healing of gastric and duodenal peptic ulcers; healing of a wound, ulceration, or any lesion characterized by destruction of the parenchyma and/or underlying stroma of any organ or tissue in which normal healing must proceed through granulation tissue, necessitating the influx of chronic inflammatory cells.
  • an immunocompromised host e.g. a patient on chronic high dose steroids, following cancer chemotherapy, suffering from AIDS or leukemia
  • infectious organisms or non-infectious causes such as, meningitis, encephalitis, uveitis, cellulitis, pneumonitis, pleuritis, pneumonia, pericarditis, myocarditis, endocarditis, sinusitis, pharyngitis, retinitis, otitis, esophagitis, gastritis, acute infectious enteritis, ascending cholangitis, hepatitis, pyelonephritis, cystitis, urethritis, stasis ulcers and stasis dermatitis, abscess formation in any tissue due to infections, including those arising from pneumococci, Haemophilus influenzae B, N. meningitides b and Escherichia coli, group B Streptococcus, St
  • the agents capable of modulating transendothelial leukocyte migration, their analogs, binding partner(s) or other ligands or agents exhibiting either mimicry or antagonism to the agents may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient having a tissue infection or other pathological derangement such as immune system dysfunction involving leukocytes, including neutrophils, monocytes and lymphocytes, for the treatment thereof.
  • a variety of administrative techniques may be utilized, among them topical applications as in ointments or on surgical and other topical appliances such as, surgical sponges, bandages, gauze pads, and the like, as well as orally active formulations.
  • compositions may be administered by parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, including delivery in an irrigation fluid used to wash body wound areas, catheterizations and the like. Average quantities of the agents may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • compositions useful in practicing the therapeutic methods of this invention may include, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a transendothelial migration modulator (agent)/modulator (agent) antagonist, or analog thereof, as described herein as an active ingredient.
  • Particular therapeutic compositions may further include an effective amount of the agent/agent antagonist or analog thereof, and one or more of the following active ingredients: an antibiotic, a steroid.
  • Exemplary formulations are presented in Example 12, later on herein.
  • the invention further includes a method for detecting idiopathic or known stimuli on the basis of their ability to modulate transendothelial migration via PECAM.
  • invasive stimuli could be identified and detected by their ability to either stimulate or suppress TEM by cellular agonists such as leukocytes.
  • these agonists, one of the agents of the invention, or binding partners thereto could be utilized in a variety of diagnostic protocols.
  • the noted materials may be labeled with a detectible label and employed in an assay such as the transmigration assay of the present invention, for example, to monitor or determine patient status, or to investigate and evaluate possible new drugs.
  • Exemplary diagnostic protocols are set forth below by way of illustration and not limitation. These protocols extend from the direct examination of a particular sample to the preparation and comparison of plural samples and corresponding controls and the correlation of the results as between the former and the latter, respectively, and in direct comparison with each other. In this way, direct diagnosis, as well as monitoring and drug discovery are contemplated and covered.
  • a first protocol relates broadly to a method for measuring transendothelial migration of leukocytes, comprising:
  • Step B. incubating the biological sample of Step B. with said test substrate for a period of time sufficient for the transmigration of said leukocytes to occur;
  • Step D examining the incubated material of Step C. by counting the position and number of said leukocytes adhering to the intercellular regions in said test substrate.
  • a second protocol covers a method for measuring transendothelial migration in a mammal and the role of PECAM therein, and comprises: A. providing a test substrate from a colony of human endothelial cells;
  • Step C incubating an aliquot portion of the biological sample of Step B. with said test substrate for a period of time sufficient for the transmigration of said leukocytes to occur in the absence of PECAM antagonists;
  • Step D preparing a control by incubating a further aliquot portion of the biological sample of Step B. with said test substrate and with an agent capable of modulating the transendothelial migration of leukocytes, said agent selected from the group consisting of antibodies to PECAM, antagonists of PECAM activity, recombinant human PECAM-1 (rhPECAM-l), agonists of PECAM activity, and active fragments thereof, for a period of time equal to that of Step C; and E. measuring said PECAM function in terms of transendothelial migration of said leukocytes by examining the incubated material of Step C. and comparing the results to the incubated material of Step D.
  • an agent capable of modulating the transendothelial migration of leukocytes said agent selected from the group consisting of antibodies to PECAM, antagonists of PECAM activity, recombinant human PECAM-1 (rhPECAM-l), agonists of PECAM activity, and active fragments thereof, for a period of time equal to that of Step C;
  • a third protocol extends to a method for detecting circulating modulators of PECAM function in a mammal, and comprises:
  • preparing at least one control by incubating a further aliquot portion of leukocytes from the test mammal or another mammal with said test substrate and with and/or without an agent capable of modulating the transendothelial migration of leukocytes, said agent selected from the group consisting of antibodies to PECAM, antagonists of PECAM activity, recombinant human PECAM-1 (rhPECAM-l), agonists of PECAM activity, and active fragments thereof, for a period of time equal to that of Step C; and
  • well known immunological procedures that are useful herein may utilize either the cellular agonists, the present agents or their respective antagonists or other binding partners labeled with a detectable label.
  • the cellular agonist or TEM modulator forms a complex with a binding partner and one member of the complex is labeled with a detectable label.
  • the fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine and auramine.
  • An example of a conjugate would be fluorescent anti-PECAM antibody, prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the cellular agonist or TEM modulator or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from ⁇ , 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, *Tc, In, 125 I, 131 I, and ,86 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • a particular assay system developed and utilized in accordance with the present invention is known as a receptor assay.
  • the material to be assayed is appropriately labeled and then certain cellular or solid phase biochemical targets are incubated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds specifically to the cell receptors. In this way, differences in affinity between materials can be ascertained.
  • a material to which the molecule in the test sample to be detected and assayed binds is attached to a solid surface, the test sample applied, and after appropriate incubation and washing, the amount of that molecule bound is determined by adding a labeled detector and quantitating with reference to a known standard.
  • a purified quantity of either a cellular agonist such as leukocytes or a TEM modulator may be radiolabeled, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled material, whether cellular agonist or TEM modulator, and HEC cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, soiubiiized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • kits suitable for use by a medical specialist may be prepared to measure the extent of transendothelial leukocyte migration in a particular mammalian host.
  • one class of such kits will contain at least the labeled TEM modulator or respective binding partner, for instance an antagonist specific thereto, a predetermined standard with which to calibrate and evaluate the response, and directions, of course, depending upon the method selected.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, instructions for the labeling of the patient's leukocytes, etc.
  • the methods described herein may be used to test potential drugs by their ability to interact with the cellular agonists and/or the TEM modulators, to exert an effect on transendothelial migration and concomitant inflammation.
  • the transendothelial migration of certain cells may be measured and monitored, to determine the likelihood that a patient may respond to PECAM therapy, or to test potential drugs useful to control inflammation by modulating TEM.
  • a particular method for the measurement of TEM is disclosed herein that offers a precise quantitation that is achieved by the visual technique described above and illustrated in the Examples appearing later on herein.
  • the illustrated assay is an in vitro assay of transmigration of human leukocytes across human endothelial cell monolayers, that has been developed to reflect many of the conditions present in vivo.
  • the endothelial cells in this model are phenotypically similar to endothelial cells lining blood vessels in the body, and express PECAM-1 at the junctions between them under resting as well as cytokine-activated conditions.
  • a corresponding assay of lymphocyte transmigration across HEC may be performed employing an appropriate lymphocyte cellular model.
  • activated T cells and cells such taken from myeloid and lymphoid tumor cell lines (see Ohto et al., 1985; Goyert et al., 1986) are known to express PECAM on their surfaces, and would serve in this capacity.
  • Contrast Optics directly visualizing the individual HEC monolayers and counting the percentage of leukocytes on top of, stuck within, or transmigrated through the HEC monolayer.
  • the monolayers are washed with EGTA, then DPBS, then stained with silver nitrate to outline of the HEC junctions. This makes it even easier to define the plane of HEC monolayer and to discern which leukocytes are trapped between HEC.
  • the monolayer is then fixed in 10% neutral buffered formalin or 2% glutaraldehyde in cacodylate buffer and stained with Wright-Giemsa stain.
  • the Hoffman optics make it possible to discern precisely the spatial orientation of the leukocytes with respect to the HEC monolayer.
  • Leukocytes above and below the HEC monolayer are distinguished, for example, by any of three procedures described in the Experimental Methods, and are counted in several random fields (for larger culture dishes) or in the central field in each well of a 96- well culture plate (for reproducibility). Data are expressed as the percentage of leukocytes below the HEC monolayer.
  • the above assay of the present invention is capable of automated performance.
  • the IgG coated sheep erythrocytes (E-IgG) described above in the resetting assay could be labeled with a fluorescent tag (e.g. Carboxyfluorescein succinimidyl ester (CFSE)) and fluorescence retained by the monolayer is proportional to the number of E-IgG rosetted, i.e., the number of apical leukocytes remaining. Fluorescence is read automatically on all the wells in the culture plate by means of a standard fluorescence plate reader (Millipore Cytofluor 2300).
  • CFSE Carboxyfluorescein succinimidyl ester
  • the E-IgG are prelabeled with a radioactive tag such as 51 Cr or "'Indium oxyquinoline (Muller and Weigl, 1992) and the retained radioactivity is proportional to the number of E-IgG rosetted, i.e. , the number of apical leukocytes remaining. Radioactivity is assessed by means of a gamma counter, either by removing the cultures physically into tubes for counts or using a gamma counter adapted for directly reading from 96- well tissue culture plates.
  • a radioactive tag such as 51 Cr or "'Indium oxyquinoline (Muller and Weigl, 1992) and the retained radioactivity is proportional to the number of E-IgG rosetted, i.e. , the number of apical leukocytes remaining. Radioactivity is assessed by means of a gamma counter, either by removing the cultures physically into tubes for counts or using a gamma counter adapted for directly reading from 96- well tissue culture plates.
  • the culture is lysed hypotonically or in nonionic detergent, and retained E-IgG are quantitated by assay of erythrocyte-specific markers, such as hemoglobin by a direct chemical measurement or Band 3 by an immunoassay.
  • Apical leukocytes may be assessed by their ability to bind non-permanent markers other than E-IgG. These could include, but are not limited to: a) polystyrene latex beads tagged with a fluorescent or radioactive label and conjugated to antibodies or ligands that will specifically bind to one or more of the leukocyte classes-monocytes, neutrophils, lymphocytes.
  • the antibodies or ligands that could be used in the assay include, but are not limited to: anti-CD45 for all classes of leukocytes, anti-CD 14 for monocytes or PMN, anti-CD3 for T lymphocytes, anti-CD4 for CD4+ T cells, anti-CD8 for CD8+ T cells, anti-CD16 for monocytes, PMN, and natural killer cells, anti- human IgG for B cells, etc.
  • the automated assay is carried out as described above for fluorescently or radioactively labeled E-IgG.
  • HEC Human umbilical vein endothelial cells
  • TNF ⁇ recombinant human TNF ⁇ (10 ⁇ g/ml in culture medium) for the times indicated in the figure legends.
  • TNF ⁇ induces the expression of endothelial cell adhesion molecules like E-selectin (Bevilacqua et al.1987) and VCAM-1 (Osborn et al.1989), which are not otherwise expressed by HEC in this culture system (Muller and Weigl, 1992).
  • PBMC peripheral blood mononuclear cells
  • PMN polymorphonuclear leukocytes
  • Hybridoma hec7 an IgG2a that binds to human PECAM- 1 was raised as described (Muller et al., 1989). Hybridoma hec7 was deposited with the American Type Culture Collection, Rockville, MD, on December 23, 1992 and has been assigned ATCC Accession No. HB 11227. As mentioned earlier, Hybridoma hec7.2, one of four identical viable subclones designated hec7.1 - hec7.4, was specifically deposited. MAb heel is an IgG2a raised in this laboratory from the same original fusion. It recognizes a junctionally restricted HEC integral membrane protein distinct from PECAM.
  • Clones secreting both mAb were adapted to growth in serum-free medium (Nutridoma NS, Boehringer Mannheim Biochemicals, Indianapolis, IN) and IgG selectively precipitated by ammonium sulfate. IgG was redissolved, dialyzed in PBS, and sterilized by membrane filtration. Fab and F(ab') 2 fragments were produced by incubating with immobilized papain and pepsin, respectively (Pierce Chemical Co., Rockford, IL). Undigested antibody and Fc fragments were removed by passage over Protein A- Sepharose (Pharmacia, Piscataway, NJ).
  • SDS-polyacrylamide gel electrophoresis performed under non-reducing conditions verified that the fragments were properly cut. SDS-PAGE of overloaded samples demonstrated that all IgG and active fragment samples were pure; no extraneous bands were present on Coomassie blue stain.
  • HB 180 LB-2, anti-ICAM-1, from Dr. Edward Clark, University of Washington, Seattle, and 3G8, anti-Fc7RIII, from Medarex, W. Lebanon, NH.
  • Rabbit anti-PECAM serum and preimmune serum were generated against PECAM- 1 purified from platelets (Albelda et al., 1991).
  • a cDNA encoding a form of PECAM containing the signal sequence and 5 1/2 of the 6 extracellular immunoglobulin loops was constructed from an existing full- length PECAM cDNA clone in the vector pcDM8 (Muller et al., 1992).
  • This plasmid was cut with restriction enzymes Hindlll and Nhel, the 1850 bp fragment encoding the external domain of PECAM was purified on an agarose gel, and ligated to a gel-purified 4056 bp fragment of pcDM ⁇ previously digested with Hindlll and Xbal representing the entire vector minus the multiple cloning domain from the first Xbal site to the Hindlll site.
  • COS-1 cells (American Type Culture Collection) were transiently transfected by electroporation of 10 7 cells in 0.5 ml Dulbecco's Modified Eagle's Medium (DMEM) with 20 ⁇ g plasmid in a gene pulser (Bio-Rad Laboratories, Richmond, CA) at 240mV, 960 ⁇ F, 4 mm path length cuvettes. The electroporated cells were plated in DMEM + 10% FCS overnight, passed by trypsinization the next day to help remove dead cells and replated in DMEM + 10% FCS. Expression of soluble PECAM was checked by immunoprecipitation from conditioned medium and immunofiuorescence of COS cells on day 3.
  • DMEM Dulbecco's Modified Eagle's Medium
  • An immunoaffinity matrix was made by covalently coupling hec7 mAb to Affi-gel 10 (Bio-Rad Laboratories) at a concentration of 3.8 mg/ml, according to the manufacturer's directions.
  • Conditioned medium was collected on day 5 from COS-1 cells transiently transfected with the construct described above, centrifuged free of cells, and passed over a 3 ml column of hec7-Affi-gel at 0.3 ml/min. The column was washed with 10 columns of SA buffer, 8 volumes of detergent buffer, and 20 volumes of SA buffer (Muller and Gimbrone, 1986).
  • Soluble PECAM was eluted in 5 ml of 0.05 M diethylamine pH 11.5, neutralized quickly with 1 M Tris pH 8, and dialyzed overnight against PBS. The dialysate was concentrated using a Centricon ® 30 microconcentrator (Amicon Division, Beverly, MA). Purity was assessed by SDS-PAGE followed by Coomassie blue or silver nitrate stain. Protein concentration was determined using the BCA assay (Pierce Chemicals). Forty micrograms of recombinant soluble PECAM were purified from approximately 350 ml of conditioned medium.
  • PBMC or PMN were resuspended to 2 or lxlOVml, respectively, in warm medium 199 (M199) or complete culture medium (20% normal human serum in M199) and allowed to settle on confluent monolayers of HEC grown in 96-well trays on hydrated collagen gels.
  • M199 warm medium 199
  • complete culture medium (20% normal human serum in M199)
  • anti- PECAM mAb, other anti-PECAM reagents, or control mAb were also added at the same time.
  • Transendothelial migration was allowed to proceed at 37°C, usually for one hour.
  • the fixed HEC monolayer, associated leukocytes and collagen gel were physically removed from the 96-well tray and processed for scanning EM by critical point drying and gold coating as described (Phillips and Bourinbaiar, 1992).
  • Specimens were examined on an ETEC Scanning Electron Microscope. For quantitative evaluation, each specimen was centered in the beam at low power (ca. 20X magnification), then monocytes were counted in at least ten random, non-overlapping 1,000X fields (each field approximately 10,323 ⁇ m 2 ) at or near the center.
  • Apical monocytes were observed as large, mostly spread cells with at least one pseudopod. Cells resembling lymphocytes were seen only rarely on the apical surface in the one hour and three hour specimens. At five hours apically adherent lymphocytes were observed at the same density (approximately two per field) in control and anti-PECAM treated specimens, and were not counted. Data are expressed as mean ⁇ standard deviation number of apical monocytes per 1000X field.
  • Chemotaxis assay Hydrated collagen gels identical to those used for culture were impregnated with chemoattractant by incubation with 200 ⁇ l of chemoattractant solution overnight at 37° in the tissue culture incubator. Control experiments using fluorescently labeled IgG demonstrated that equilibration occurred within six hours (t -45 min) for both accumulation within and elution from the gel. Such gels were washed three times with M199. Leukocytes resuspended to lOVml in ml99 plus 0.1% human serum albumin were added to the wells (100 ⁇ l/well), and migration into the gels was allowed to proceed for one hour.
  • PBMC isolated from normal donors were incubated for 20 min at 4°C with anli- PECAM-1 mAb hec7 (Muller et al., 1989) or isotype matched control mAb, washed free of unbound antibody, and added to HEC monolayers in the standard transmigration assay (Muller and Wiegl, 1992). As previously reported (Muller and Weigl, 1992), after 40 to 60 minutes of incubation, all of the monocytes (Mo) in the control samples had transmigrated the HEC monolayer, while lymphocytes remained unbound or loosely attached and were easily removed by the washes in 1 mM EGTA.
  • PBMC peripheral blood monolayers
  • hec7 anti-PECAM mAb
  • control mAb control mAb
  • hec7 usually inhibited Mo transmigration by 70 to 90%.
  • the same results were obtained when the transmigration assay was carried out in the continuous presence of antibodies (Fig. 3).
  • Previous results have demonstrated that hec7 mAb added to HEC monolayers that have already achieved confluence does not disrupt junctions. Therefore, it is presumed that the effect in this experiment of the mAb continuously present is solely on the Mo.
  • Monoclonal antibody hec7 at 10 to 20 ⁇ g/ml was as effective as a 1:100 dilution of rabbit anti-PECAM serum at blocking TEM (Fig. 4).
  • Anti-PECAM mAb hec7 blocked TEM significantly at doses as low as 1 ⁇ g/ml, with maximum effect seen at around 10 ⁇ g/ml.
  • the effect of hec7 was not mediated via an effect on the monocyte Fc receptor.
  • Fab [and F(ab') 2 fragments, data not shown] fragments of hec7 also demonstrated significant inhibitory activity in the 1 to 10 ⁇ g/ml range (Fig. 5).
  • Anti-PECAM mAb does not affect chemotaxis
  • hec7 monoclonal antibody bound to PECAM in the HEC junctions blocks monocyte transmigration Greater than 85% of the PECAM on cultured HEC is concentrated in the intercellular junctions (Muller 1989). However, in this location it is poorly, if at all accessible to hec7 mAb applied to the apical surface of live, confluent monolayers. Indeed, hec7 and several other mAb recognizing membrane proteins on the endothelial cell surface (including ICAM-1) failed to block transmigration when HEC monolayers were preincubated with these mAb for 1 hr. at 37 ° C, then washed free of unbound antibody prior to addition of the PBMC (Data not shown.)
  • heel is a mAb raised in this laboratory that recognizes a novel integral membrane protein of 130 kD that, like PECAM-1, is enriched in the junctions of HEC, but unlike PECAM, is not expressed on leukocytes or platelets. Furthermore, heel is an isotype match for hec7.
  • Fab fragments of heel also accumulated in HEC intercellular junctions (Fig. 9b).
  • heel Fab pretreatment had no effect on transmigration of untreated monocytes (Fig. 10, right group "heel Fab").
  • hec7 IgG was added to monocytes exposed to hec7 Fab pre-treated monolayers (Fig. 10, filled bar, hec7 Fab group) than was achieved by adding this anti-PECAM reagent to either the monocytes (Fig. 10, filled bars, control and heel Fab groups) or the HEC (Fig. 10, open bar, hec7 Fab group) alone.
  • hec7 is blocking an interaction in which PECAM on the monocyte is binding in a homophilic manner with PECAM on the endothelial cell.
  • the blockade of transmigration by hec7 Fab is not due to Fab leaking out of the HEC monolayer or subendothelial collagen and binding to monocyte PECAM.
  • Fab-pretreated HEC cultures were processed in an identical manner to those used in the experiments of Fig. 10. Supernatant was collected from these cultures after an hour at 37°C and used to resuspend freshly isolated PBMC, which were then subjected to the transmigration assay. Neither supernate from control or hec7 Fab treated cultures inhibited transmigration of Mo (96.8 + 2.6% and 97.6 ⁇ 2.3% transmigration, respectively).
  • Recombinant soluble PECAM blocks transendothelial migration of leukocytes If hec7 were interfering with transmigration by binding to monocyte and/or endothelial cell PECAM and blocking either or both sides of a homophilic adhesion, it might be possible to use a soluble form of the molecule to compete with endothelial PECAM for the Mo and thus block transmigration.
  • a soluble recombinant form of PECAM- 1 was prepared that was truncated in the middle of the sixth immunoglobulin domain. This construct, expressed in COS cells is secreted into the medium and migrates in SDS-PAGE gels at the predicted Mr of 90 kD (Fig. 11). Soluble PECAM was purified on a hec7-Sepharose affinity column from culture medium conditioned by COS cells transiently transfected with this construct. The purified material runs as a single band at the expected position on SDS-PAGE.
  • Anti-PECAM mAb blocks transmigration of Mo across cytokine activated HEC monolayers.
  • the in vitro transmigration assay used in this study employs HEC monolayers that are not activated by exogenous cytokines nor endotoxin.
  • the endothelial cells do not express cytokine inducible adhesion molecules like VCAM-1 or E-selectin and have basal levels of ICAM-1 (Muller and Weigl, 1992).
  • cytokine inducible adhesion molecules like VCAM-1 or E-selectin and have basal levels of ICAM-1 (Muller and Weigl, 1992).
  • transmigration of Mo is even more rapid than under basal conditions, with 100% of Mo entering the subendothelial collagen within 20 to 30 min (unpublished data). It was therefore of interest to determine whether hec7 would have as dramatic effect on monocytes transmigrating under these conditions. As can be seen in Figure 12, it does.
  • Neutrophils also bear surface PECAM (Ohto et al., 1985; Goyert et al., 1986; Stockinger et al., 1990), although hec7 does not bind to the form displayed by PMN as well as it binds HEC and Mo (unpublished observations). Neutrophils do not migrate readily across resting HEC monolayers in this system (Muller and Weigl, 1992). However, they do transmigrate cytokine-activated monolayers readily (unpublished). After 1 hour 97% of PMNs in the presence of control antibody had migrated below the TNF ⁇ -activated HEC monolayer.
  • hec7 MAb blocked transmigration by about 40%, consistent with its relatively weak binding to the neutrophil form of this molecule.
  • a polyclonal rabbit antiserum against PECAM and soluble recombinant PECAM at 10 ⁇ g/ml blocked transmigration down to below 20% of control levels (Fig. 13).
  • PECAM apparently plays a similar role in the transmigration of neutrophils as it does for monocytes.
  • mice of the CD2F, strain weighing approximately 20 g. were purchased from Charles River Laboratories (Boston, MA) and housed at The Rockefeller University Laboratory Animal Research Center.
  • Female mice of the AKR/J strain were purchased from Jackson Laboratories (Bar Harbor, ME) and housed at the Boston University School of Medicine Laboratory Animal Science Center. All animal procedures had been approved by the Rockefeller University and Boston University School of Medicine IACUCs. Animals were handled according to guidelines set forth in the "Guide for the Care and Use of Laboratory Animals" and the "Animal Welfare Act. " Mice were housed together in standard cages and allowed free access to mouse chow and water.
  • Monoclonal antibody (mAb) 2H8 hamster anti-murine PECAM-1 was produced as described (Bogen et al. 1992) and purified by HPLC using a JT Baker ABx semi-preparative column, loading in 25 mM 2-[N- Morpholino]ethanesulfonic acid (MES), pH 5.7, and eluting with a linear 10%- 60% gradient of 500 mM NHjSO + 5 mM KH 2 PO 4 , pH 6.7, according to the manufacturer's recommendations. Purification of the 2H8 mAb under denaturing conditions resulted in an antibody preparation that was less effective in vivo.
  • MES 2-[N- Morpholino]ethanesulfonic acid
  • the 2H8 mAb was negative for endotoxin by the Limulus amebocyte lysate assay (Sigma, St. Louis, MO).
  • the hamster anti-murine CD 18 mAb 2E6 was originally raised at Rockefeller University (Metlay et al. 1990) and was donated by Endogen, Inc. (Boston, MA).
  • Hybridoma lines producing mAb 5C6 (rat anti-murine CD lib) (Rosen and Gordon 1987) were generously provided by Dr. Hugh Rosen (Merck, Sharp & Dohme Research Laboratories, Rahway, NJ); IgG was purified from cell supernatant as previously described (Muller et al. 1993). Normal hamster IgG was purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA). All mAb were demonstrated to recognize their specific antigens by flow cytometry and/or immunohistochemistry.
  • DPBS Dulbecco's phosphate buffered saline
  • mice were sacrificed by exposure to CC Peritoneal cells were recovered into tubes on ice by lavage with 5 ml of divalent cation-free Hanks' Balanced Salt Solution (HBSS, Gibco, Grand Island, NY) using standard techniques (Muller et al. 1980). Heparinized blood was coUected by cardiac puncture or from the retro-orbital sinus. Peritoneal cell counts and peripheral white blood cell counts were performed (employing Unopette erythrocyte lysis kits, Fisher Scientific, Pittsburgh, PA) on samples from each mouse using a hemacytometer. Differential counts were performed on Wright/Giemsa stained cytospin preparations (for peritoneal cells) and peripheral blood smears.
  • HBSS Hanks' Balanced Salt Solution
  • the abdominal and thoracic cavities were inspected for signs of gross pathology.
  • the liver and spleen were excised intact and weighed. These organs, along with the majority of the small and large bowel were fixed in neutral buffered formalin. Representative regions were embedded in paraffin or methyl methacrylate. Sections were cut and stained with hematoxylin and eosin (H&E).
  • H&E hematoxylin and eosin
  • PECAM- 1 is required for the migration of monocytes and neutrophils across resting and cytokine-activated HEC in a quantitative in vitro assay of transmigration (MuUer et al., 1993), and prompted the observation and conclusions that PECAM is a central actor in inflammation and other conditions where leukocyte migration occurs in response to invasive stimuU such as infection.
  • Monoclonal antibody against murine PECAM- 1 blocks acute inflammation.
  • PECAM- 1 plays a critical role for acute inflammation in vivo
  • the hamster anti-murine PECAM- 1 mAb 2H8 (Bogen et al., 1992) or appropriate positive or negative control mAb, was administered intravenously (i.v.), four hours prior to the intraperitoneal (i.p.) injection of thioglycoUate broth ( Figures 14 and 15.)
  • intraperitoneal thioglycoUate induces an influx of neutrophils into the peritoneal cavity within the first two hours (Lewinsohn et al., 1987; Watson et al., 1991).
  • Figures 14 and 15 separately report data on the PMN exudate and the total exudate cells (including lymphocytes and macrophages).
  • the decrease in PMN accumulation produced by the anti-PECAM-1 mAb is particularly dramatic, since these ceUs are not normally resident in the peritoneal cavity. Additional experiments revealed that significant suppression of inflammation by mAb 2H8 was achieved at doses as low as 50 ⁇ g/mouse (the lowest dose tested-Fig. 14), and suppression was maintained for at least 48 hours (not shown).
  • Anti-PECAM- 1 mAb appears to arrest emigration of adherent leukocytes Administration of these antibodies did not lower the circulating leukocyte count. In fact, at the time of sacrifice, there was a granulocytosis in those mice that had received anti-PECAM- 1 mAb (Table I), consistent with the hypothesis that PMN were being recruited into the circulation, but blocked from emigrating to the site of inflammation. Histologic examination revealed no abnormal accumulations of leukocytes in the spleens or livers (not shown). Absolute platelet counts were not performed, but no difference in platelet numbers or appearance on peripheral blood smears was seen among the experimental groups.
  • Anti-PECAM- 1 mAb blocks emigration of mononuclear cells
  • Significant numbers of mononuclear cells as well as PMN are recruited to the peritoneal cavity by 20 hours after thioglycoUate injection.
  • Figures 14 and 15 demonstrate that the increase in peritoneal exudate cells is not accounted for by the increase in PMN alone.
  • Anti-PECAM- 1 mAb 2H8 also blocked the influx of mononuclear cells into the peritoneum (Fig. 15, right).
  • the mesenteric lymph nodes draining the peritoneal cavity were examined.
  • the peritoneal cavity is drained by, among other lymphatic beds, the mesenteric plexus.
  • Peritoneal mononuclear cells in the afferent lymphatics would enter draining lymph nodes via the subcapsular sinus.
  • Examination of mesenteric nodes revealed large numbers of mononuclear cells entering via the subcapsular sinus in mice stimulated with thioglycoUate, but relatively few in the subcapsular sinuses of mice given anti-PECAM- 1 mAb prior to thioglycoUate (Figure 17).
  • the paucity of mononuclear cells in the subcapsular sinus is a direct reflection of the block of mononuclear cell entry into the peritoneal cavity. These data are in agreement with those of Figures 14 and 15, and selectively represent the mononuclear cell fraction. PMN do not recirculate into the draining lymphatics in general and were not seen in the subcapsular sinuses of the lymph nodes of these mice.
  • the thioglycollate-elicited peritoneal exudate cell count was lower for anti-PECAM- 1 treated mice than for control mice not stimulated with thioglycoUate ( Figure 14 and Figure 15, right). This suggested that PECAM- 1 may be required for the constitutive trafficking of mononuclear cells through the peritoneum as well as for the thioglycollate-elicited emigration.
  • anti-PECAM- 1 mAb or soluble recombinant PECAM- 1 did not affect attachment, but blocked transmigration of monocytes and PMN (Muller et al., 1993), which remained tightly adherent to the apical surface over the intercellular junctions, the site at which endothelial PECAM- 1 is concentrated (Muller et al., 1989).
  • the function of PECAM- 1 in transendothelial migration was distal to the selectin-mediated rolling and the ⁇ 2 integrin-mediated tight adhesion to the apical surface of the venular endothelium.
  • leukocyte transmigration could be blocked equaUy well by treating either the leukocytes or the endothelial cells with anti-PECAM- 1 reagents.
  • PECAM- 1 may mediate a function common to all leukocyte types that is necessary for the process of transendothelial migration.
  • the inhibition was effected at a low dose of anti-PECAM- 1 mAb (50 ⁇ g/mouse in certain experiments) and lasted up to 48 hours following a single injection.
  • Example 9 The murine model of peritonitis that was presented in Example 9 confirmed the role of PECAM- 1/CD31 in inflammation that demonstrated in Examples 1-8.
  • the monoclonal antibody specific for murine PECAM- 1 injected intravenously four hours prior to the intraperitoneal injection of thioglycoUate broth blocked leukocyte emigration into the peritoneal cavity for up to 48 hours. This block was particularly evident for neutrophils.
  • Vaporciyan, et al. (1993) Science, 262:1580-1582) further corroborate the premise and conclusions of the present invention.
  • Vaporciyan et al. studied PECAM- 1 activity in three distinct models of inflammation, and found that in all three models, PECAM demonstrated an active role in the promotion of neutrophil migration.
  • the authors prepared a rabbit polyclonal antibody reactive with human PECAM- 1 and cross-reactive with rat PECAM- 1, and after establishing its specificity, tested its activity in two compartments in rats, in its effect on neutrophil accumulation induced by glycogen introduced into the peritoneal cavity, and the deposition of immunoglobulin G (IgG) immune complexes in the lung, and on human skin transplanted onto mice with severe combined immunodeficiency disease (SCID).
  • the anti-PECAM-1 antibody was injected intravenously into the rats at the time of glycogen instillation and periodically thereafter, and blocked glycogen-induced peritoneal neutrophil accumulation by 75%.
  • the antibody was infused intravenously at 30 minute intervals commencing 2.5 hours after initiation of immune complex injury, and again, a 75% reduction in neutrophil recruitment was observed.
  • the skin grafts of the third model were challenged with TNF- ⁇ four weeks after transplantation, and those grafts receiving the antibody exhibited a reduction in neutrophil recruitment and movement. The results of these tests both corroborate the present findings regarding PECAM's activity and further confirm the therapeutic utility of the modulators of the present invention.
  • polypeptides corresponding to the ligand binding domain of PECAM- 1 or, for example, a 210 amino acid peptide encompassing Domains 1 and 2 of PECAM-1 would be administered parenterally to a patient afflicted with a flare-up of a chronic inflammatory condition (e.g. Systemic Lupus Erythematosis).
  • a chronic inflammatory condition e.g. Systemic Lupus Erythematosis
  • the soluble peptide would interfere with PECAM-PECAM interactions of leukocytes and endothelial cells, such that transendothelial migration is inhibited and the leukocytes, unable to cross the endothelial junctions into the sites of inflammation, return to the circulation after a brief period of arrest on the apical surface of the endothelium (just as occurred when anti-PECAM mAb was administered parenterally in a murine model of inflammation, Figures 14-16). It would be expected that the deleterious effects of the inflammatory flare-up (e.g. renal damage) would be ameliorated. This could be monitored, for example, in this instance, by standard clinical measurements of renal function.
  • the inflammatory flare-up e.g. renal damage
  • Changes in secondary structure of adhesion molecules have been demonstrated in some cases to be crucial for these molecules to function. Therefore, if such a situation is found for PECAM- 1, molecules found to inhibit or promote the formation or retention of the appropriate structural changes may be used to block or augment PECAM function, respectively.
  • Such blocking or augmenting molecules may be peptides, small organic molecules, and inorganic molecules, e.g. metal ions and complexes thereof.
  • the aforementioned are nonlimiting examples of how molecules other than anti-PECAM antibodies or full-length soluble PECAM- 1 may be used in accordance with the present invention.
  • Intravenous Formulation I Ingredient cefotaxime monoclonal antibody hec7 dextrose USP sodium bisulfite USP edetate disodium USP water for injection q.s.a.d.
  • the present invention is applicable to the treatment of a variety of inflammatory disease states including infectious diseases where active infection exists at any body site, such as in the instance of meningitis.
  • conditions such as secondary inflammations whether acute or chronic, that may occur at a site of antigen deposition that is secondary to a primary infection at a distant body site
  • exemplary specific conditions would also include meningitis, as weU as encephalitis, arthritis, uveitis, colitis, such as inflammatory bowel/Crohn's disease, glomerulonephritis, dermatitis, and psoriasis.
  • inflammation that results from alterations in leukocyte movement during infection such as adult respiratory distress syndrome associated with sepsis.
  • inflammatory disease states include immune disorders and conditions involving T-cell and/or macrophage attachment recognition; such as, acute and delayed hypersensitivity, graft vs. host disease; primary auto-immune conditions such as pernicious anemia; infection related auto-immune conditions such as Type I diabetes mellitus; flares during rheumatoid arthritis; diseases that involve leukocyte diapedesis, such as multiple sclerosis; antigen-antibody complex mediated diseases including certain of the secondary infection states listed above; and transplant rejection. Inflammation due to toxic shock or trauma such as adult respiratory distress syndrome and reperfusion injury; and that which is due to tumorous conditions such as leukocyte dyscrasias and metastasis, is likewise included within the scope hereof.
  • primary auto-immune conditions such as pernicious anemia
  • infection related auto-immune conditions such as Type I diabetes mellitus
  • flares during rheumatoid arthritis diseases that involve leukocyte diapedesis, such as multiple s
  • the present invention is applicable to the inhibition of leukocyte transmigration in the instances of non-disease states and in particular, for diagnostic and therapeutic purposes; such as to prevent the ingress of leukocytes during the introduction of a dye or image enhancer into tissue, or to allow the selective entry of a therapeutic drug in the instance of chemotherapy; or to enhance the harvesting of leukocytes from patients.
  • the following is a listing of certain of the publications referred to in abbreviated fashion in the foregoing specification.
  • EndoCAM a novel endothelial cell-cell adhesion molecule. J. Cell Biol , 110:1227-1237.
  • PECAM- 1 endoCAM/CD31
  • the circulating phagocyte reflects the in vivo state of immune defense. Curr. Op. Inf. Dis. , 5:389-398.
  • Neutrophil-activating protein 1/Interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD1 lb/CD 18 on human neutrophils. J. Exp. Med. , 171:1155-1162.
  • Lymphocyte homing receptors Cell, 44:673-680.
  • Gallatin W.M., Weissman, I.L., and Butcher, E.C. (1983). A cell-surface molecule involved in organ-specific homing of lymphocytes. Nature, 304:30-34.
  • Leukocytes roll on a selectin at physiologic flow rates: Distinction from and prerequisite for adhesion through integrins. Cell, 65:859-873.
  • Lectin-like cell adhesion molecule 1 mediates leukocyte rolUng in mesenteric venules in vivo. Blood, 77:2553-2555.
  • Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CD3 (CD 1 IB/CD 18, Mac-1, alpha m beta 2) on human neutrophils. J. Exp. Med. , 173:1493-1500.
  • PECAM-1 an adhesion molecule at the junctions of endothelial cells.
  • Mononuclear Phagocytes Biology of Monocytes and Macrophages, R. van Furth, ed. (Kluwer Academic Publishers),
  • Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination. J. Cell Biol, 103:2389-2404.
  • PECAM-1 is required for transendothelial migration of leukocytes. J. Exp. Med. , 178:449-460. Newman, P.J., Berndt, M.C., Gorski, J., White II, G.C., Lyman, S., Paddock, C, and Muller, W.A. (1990). PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science, 247: 1219-1222.
  • a novel leukocyte differentiation antigen Two monoclonal antibodies TM2 and TM3 define a 120-kd molecule present on neutrophils, monocytes, platelets, and activated lymphoblasts. Blood, 66:873-881.
  • vascular cell adhesion molecule 1 a cytokine-induced endothelial protein that binds to lymphocytes.
  • Leukocyte adhesion molecule- 1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion. J. Immunol , 147:2565-2573.
  • Monocyte attachment to activated human vascular endothelium in vitro is mediated by leukocyte adhesion molecule- 1 (L-selectin) under nonstatic conditions. J. Exp. Med. , 175: 1789-1792.
  • CD31 expressed on distinctive T cell subsets is a preferential amplifier of betal integrin-mediated adhesion. J. Exp. Med. , 176:245-253.

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Abstract

La migration transendothéliale des leucocytes ou autres globules lors d'une inflammation aiguë et/ou chronique peut être inhibée par l'administration de réactif anti-PECAM-1. Notamment, la migration transendothéliale semble être induite par l'activité ds molécules d'adhésion des cellules endothéliales des thrombocytes (PECAM), étant donné que l'inhibition de la migration transendothéliale (TEM) des granulocytes neutrophiles et des monocytes se produit en présence des PECAM solubles recombinées ou d'un anticorps anti-PECAM monoclonal. L'invention concerne également des procédés de régulation de la migration transendothéliale et des agents correspondants, ainsi que des procédés de diagnostic et des méthodes de recherche de médicaments. L'inhibition de cette migration transendothéliale pourra jouer un rôle dans la prévention et/ou la réduction de l'inflammation provoquant la lésion d'organes, et ce procédé peut être notamment utile dans le traitement d'inflammations chroniques et aiguës.
EP94907806A 1993-01-12 1994-01-12 Procede de modulation de la migration transendotheliale des cellules favorisant l'inflammation et procedes connexes de mesure de cette migration Withdrawn EP0675735A1 (fr)

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GB2294321A (en) * 1994-10-19 1996-04-24 Yamanouchi Research Inst Method of screening for CD31 interaction inhibitors
US10539487B2 (en) 2010-03-04 2020-01-21 Ventana Medical Systems, Inc. Systems and methods for monitoring tissue sample processing
US10126216B2 (en) 2011-02-17 2018-11-13 Ventana Medical Systems, Inc. Method for tissue sample fixation
AU2011222501B2 (en) 2010-03-04 2014-05-01 Ventana Medical Systems, Inc. Processing system for processing specimens using acoustic energy
US9901625B2 (en) 2011-10-15 2018-02-27 University Of Maryland, College Park Methods of regulating uptake and transcellular transport of leukocytes and therapeutics

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9415641A1 *

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KR960700071A (ko) 1996-01-19
AU6799898A (en) 1998-07-16
WO1994015641A1 (fr) 1994-07-21
CA2153776A1 (fr) 1994-07-21
JPH08505855A (ja) 1996-06-25
HUT73462A (en) 1996-08-28
AU6122894A (en) 1994-08-15
HU9502116D0 (en) 1995-09-28

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