CA2037345A1 - Endothelial cell-monocyte adhesion molecule - Google Patents

Abstract

39. PATENT

ABSTRACT

Receptor isolated and substantially purified from endothelial cells which is specific for a ligand present on monocytes.

Description

Express Mail No. FB389479200 US
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1. PATENT

ENDOTHELIAL CELL MONOCYTE ADHESION MOLECULE

This work was supported by a Grant from the National Institutes of Health. The United States Government may retain certain rights of this invention.

BACKGROUND OF THE INVENTION

5Field of The Invention The present invention relates to receptor molecules which are involved in the adhesion of monocytes to endothelial cells.

Related Art Inflammation occurs as a consequence of tissue damage. This tissue damage can result from microbial invasion, auto-immune processes, tissue infection, allograft rejection, or such harmful external influences as heat, cold, radiant energy, electrical or chemical stimuli, or mechanical trauma. Whatever the causeor bodysite, the inflammatory response is quite similar, consisting of a complicated series of functional and cellular adjustments, involving the microcirculation, fluid shifts, and inflammatory leukocytes. When tlssue damage occurs, soluble chemical substances are elaborated which initlate the inflammatory response. Theinflammatory response consists of a complex series of events which include a locallzed increase in blood flow, with capillary dilation and increa$ed permeability for the fluid components of the blood; a localized exudation of fluid at the site of injury, including the proteins of plasma that normally leave the capillaries at a relatively low rate; and the exudation of leukocytes from the capillaries into the inflammatlon site. This exudate initially consists primarily of polymorphonuclear leukocytes, followed by monocytes, Iymphocytes, and plasma cells. These leukocytes produce a variety of mediators that control the extent and duration of the inflammatory response. In addition, the leukocytes have a series of receptors ;~03~3~5 2. PATENT

available to react with the various chemical mediators and proteins that are part of the inflammatory fluid. Such leukocyte receptor-mediator or protein interactions are important in controlling leukocyte function within the inflammatory site. At the cellular level, inflammation involves the adhesion of leukocytes to the endothelial wall of blood vessels and the infiltration of these leukocytes into the surrounding tissues. Thus, a major event in the infiltration of leukocytes in the inflammatory response, is adhesion of the leukocyte to receptors present on the endothelial cell surface.

A number of adhesion molecules controlling leukocyte adhesion to endothelial 1 0 cells have been described including: ELAM-1, ICAM-1, and VCAM-1. ELAM-1 has been cloned by Bevilacqua, et a/., (J. Clin.lnvest, 76:2003-, 1 985) and selectively mediates neutrophil and monocyte, but not Iymphocyte, binding. ELAM-1 is induced on human umbilical vein endothelial cells (HUVEC) by IL-1, TNF-alpha, and LPS (Pober, et a/., J. Immvnol., 137:1893-, 1986), reaching a maximal level after 2 to 4 hours and disappearing by 24 hours. An NH2 terminal lectin domain is one of the structural characteristics of ELAM-1. ELAM-1 is a member of the LEC-CAM family which include GMP-140 and LEC-CAM-1 (murine MEL-1 4 Ag, and its human homologues LAM-1, Leu8/TQ1, and DREG). All of these proteins express the N-terminal calcium-dependent carbohydrate recognition domains in which sugar moieties are important for binding (Lowe, et al., Cell, 63:475-484, 1 990).

A second inducible endothelial molecule, ICAM-1, seems to be involved in all leukocyte binding (Pober, et al., J. Immunol., 137:1893-, 1986; Dustin, et al., J.
Immunol., 1 37:245-254, 1 986). It is induced by treatment with IL-1, TNF alpha, IFN
gamma, Iymphotoxin, LPS, and phobol ester, and its expression is maintained for at least 48 hours. The ICAM-1 peptide is heavily glycosylated and it is a memberof the immunoglobulin supergene family. LFA-1, one of the integrin family, is a known ligand for ICAM-1. Anti-CD1 8 was reported to reduce monocyte adherence to unstimulated HUVEC, but had no significant effect on HUVEC pretreated with ;~03 3. PATENT
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TNF alpha (Carlos, et al., Immunol.Reviews, 114:5-28, 1990) suggesting that monocyte binding to HUVEC following treatment with TNF alpha may not be due to ICAM-1 induction.

VCAM-1, recently described by Obsorn, eta/. (Cell, 59:1203-1211,1989), is rapidly induced by IL-1 and TNF alpha, or LPS, and sustained for 48-72 hours. It has been suggested that it may play a major role in Iymphocyte and monocyte, but not neutrophil, recruitment into chronic inflammatory sites. VCAM-1 belongs structurally to a subset of the immunoglobulin supergene family and the VlA-4 integrin is known to be a ligand for VCAM-1. VLA-4 binding to VCAM-1 is independent of the VLA-4 interaction with fibronectin.

The recognition that the first step in inflammation is the adhesion of the leukocyte to the vascular endothelium has enhanced the attractiveness of therapy which would prevent this reaction. Such therapies, especially those which are based onthe endothelial receptor or ligands or inhibitors of the endothelial receptor, are particularly attractive since they are more likely to be well-tolerated by the host and, thereby, less toxic. Particularly desirable would be the use of such receptors which are specific for the various types of leukocytes. In this way, it would bepossible to selectively diagnose or treat a disorder associated with a particular type of leukocyte. Unfortunately, previously known receptors were not specific for the type of leukocytes known as monocytes and, as a consequence, it was not possible to design specific therapies which would inhibit only the interaction between a monocyte and its receptor on the endothelial cell surface. This type of diagnosis and therapy would be especially useful for those categories of inflammatory pathologies wherein tha monocyte is an etiologic agent. The presentinvention addresses this need by providing a receptor with such specificity.

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4. PATENT

SUMMARY OF THE INVENTION

Recognizing the role that leukocytes play in inflammation and the possible relationship that receptors may play in augmenting this phenomenon, the inventors developed a novel reagent for inducing a receptor on endothelial cells which is specific for monocytes. These efforts have culminated in the substantial purification and identification of an Endothelial Cell Monocyte Adhesion Molecule ~EMAM) .

This receptor, as well as ligands and inhibitors of this receptor and related fràgments, can be used diagnostically and therapeutically to block the adhesion of monocytes to the EMAM receptor. Particularly relevant is the fact that this receptor is normally present in the host such that the tikelihood of toxicity can be minimized. Another major advantage of the present invention is that it provides the art with an immunosuppressive agent which, although highiy effective, can beutilizsd at concentrations which minimize the likelihood of host toxicity.

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DESCRIPTION OF THE FIGURES

FIGURE 1: Effect of MM-LDL and IL-1 on THP-1 and HL 60 adhesion. HUVEC
were either (1) untreated(none), (2) treated for 4 hours with MM-LDL(100 flg/ml), or (3) treated with IL-1 (10 U/ml) for 4 hours. After treatment, cells were rinsed with serum free medium and the monocytes or neutrophils added for 1 hour.
Nonadherent ceils were removed by washing and adherent cells were counted.
Values represent mean +S.D.(n=3).

FI~URE 2: U937 and T cell adhesion to HUVEC. HUVEC monolayers were incubated with MM-LDL(40 ~g/ml), or LPS (10 ng/ml) for 4 hours at 37OC, washed in PBS and placed in adhesion chambers. Leukocytes suspended in PBS were injected into the chambers and allowed to remain in contact with the monolayers for 600 seconds. The number of leukocytes in contact with the monolayers was then determined, the chambers inverted for 500 seconds, and the percentage of leukocytes remaining attached determined. Values are significant (p<0.01) for both types of leukocytes. n=4 separate experiments (U937 cells), n=a (T cells untreated and MM-LDL treated), and n=5 (T cells, LPS).

FIGURE 3: Induction of adhesion molecules. HUVEC were treated with MM-LDL(100 ~g/ml), the same preparation as used as in Figure 1, and IL-1(10 U/ml) for 4 hours at 37OC. Treated cells were incubated with primary MAbs (anti ELAM-1 ;P6E2, anti VCAM-1 ;P1 B5, and anti ICAM-1 ;P3G1 ) for 1 hour and then wHh peroxidase labeled goat anti-mouse second antibody for 2 hours. .Values are O.D.from microtiter plate reader (n=5).

FIGURE 4: Effect of FN-Ab and MCP-1-Ab on monocyte adhesion. After treatment of RAEC with MM-LDL, endothelial monolayers were treated with 10 ~g/ml of Fab fragments of MCP-1-Ab or FN-Ab, and then monocytes added in the presence and absence of antibodies. Values represent mean +S.D. (n=4).

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FIGURE 5: Effect of cycloheximide and tunicamycin on MM-LDL induced monocyte binding. RAEC were pretreated with (CH+) or without (CH-) cycloheximide (1 ~g/ml) and with (TU+) or without (TU-) tunicamycin (4 ~lg/ml) for 2 hours prior to the addition of MM-LDL. Monocyte binding was determined after 4 hour MM-LDL incubation. Values represent mean +S.D. (n=4).

FIGURE 6: Effect of sugars on monocyte adhesion. RAEC were treated with MM-LDL for 4 hours, and fixed in 1% paraformaldehye for 20 min at 4C. After washing the fixed cells with PBS, the sugars (lactose-1-phosphate and maltose-1-phosphate:10~ M, N-acetylglucosamine:102 M) were added for 30 minutes.
Monocyte binding as determined in the presence of sugars. Values are mean +S.D. (n=4).

FIGURE 7: Effect of calcium and magnesium on monocyte binding. Before adding THP-1 cells to the fixed endothelial monolayer, THP-1 cells were rinsed with 1% PBS containing 1 mM of EDTA or EGTA. Monocyte binding was determined in the presence or absence of 6 mM calcium or magnesium (EGTA
experiments) or 3mM of calcium or magnesium (EDTA experiments). Values are mean ~S.D. (n=5).

FIGURE 8: Effect of H7 and HA 1004 on MM-LDL action. RAEC were pretreated with H7(100 ~M/ml) or HA 1004 (100 ~M) for 30 minutes at 37OC and then incubated with MM-LDL(2 ~.g/ml) for 4 hours at 37C. The monocyte binding assay was carried out and adherent cells counted visually. Values represent mean +S.D. n=5.

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FIGURE 9: Induction of integral membrane proteins by MM-LDL RAEC were treated with MM-LDL for 4 hours and labeled with 35S methionine for the last 30 minutes of incubation. Cells were Iysed in 1% Triton X-114 (in 10mM Tris-HCI, 150mM NaCI, pH 7.4, lmM PMSF, 20 ~,g/ml pepstatin and leupeptin). The detergent phase was subjected to TCA precipitation, analyzed by SDS PAGE, and autoradiography.

FIGURE 10: Glycopeptidase treatment of MM-LDL induced integral membrane proteins. The membrane preparation from FIGURE 9 was incubated with glycopeptidase F (2 U/ml) for 18 hours at 37OC and analyzed by SDS PAGE and 1 0 autoradiography.

FIGURE 11: Induction of integral membrane proteins by MM-LDL. Human aortic endothelial cells were treated with MM-LDL for 4 hours and labeled with 35S
methionine for the last 30 minutes of incubation. Cells were Iysed in 1% Triton X-114 (in 10mM Tris-HCI, 150mM NaCI, pH 7.4, 1mM PMSF, 20 ~g/ml pepstatin and leupeptin). The detergent phase was subjected to TCA precipitation, analyzedby SDS PAGE, and autoradiography. C=control; M=MM-LDL treated.

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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

EMAM is a receptor which is expressed by endothelial cells. EMAM can be induced by treating endothelial cells with minimally oxidized low density lipoprotein (MM-LDL). The EMAM receptor induced by MM-LDL specifically binds a ligand pr0sent on monocytes which is essentially absent from neutrophils or Iymphocytes.

According to the present invention, MM-LDL can be prepared by oxidizing LDL by such techniques as storing the LDL in a physiological buffer at refrigeration temperatures for several months or by chemical oxidation, for example, by exposure to ferrous sulfate. When chemical treatment is used to generate MM-LDL, the oxidation substance is usually used for a period from about 2 hours to about 96 hours. LDL may also be oxidized by ex,oosure to UV light (Dousset, et a/., 8iochim Biophy. Acta, 1045:21~223, 1990) or by incubation with soy bean lipoxygenase (Sparrow, et al, J. Lipid ~es., 29:745-753, 1988). Oxidation conditions can be selected to produce LDL par~icles that contain 2-5 nmoles of thiobarbituric acid reactive substance (TBARS~ per mg of cholesterol. However, having this TBARS content is a necessary but not sufficient condition for an active preparation. Further tests such as HPLC may be necessary to detect levels of particular oxidized lipids. Activity of individual preparations can be further screened by measuring ttheir ability to induce EMAM receptors on endothelial cells.

EMAM receptors can be induced on endothelial cells by exposing the cells for a period of from about 2 hours to about 8 hours using from about 1 ~g/ml to about 200 ~g/ml of MM-LDL. These incubation times and concentrations may vary depending on the source of the endothelial cells. For example, rabbit endothelial ceils are preferably treated with from about 1 ~g/ml to about 5 ~g/ml of MM-LDL,whereas human endothelial cells are preferably treated from about 50 ~.g/ml to about 150 ~,g/ml of MM-LDL. Those of skill in the art can readily ascertain the ~U373~

9. PAT~NT

appropriate exposure times and concentration of MM-LDL for endothelial cells from a particular source without undue experimentation.

The unique characteristics of the EMAM receptor include: 1) selectivity for monocyte binding, but not neutrophil or Iymphocyte binding, 2) binding is calcium dependent, but magnesium may substitute for calcium. Moreover, the evidence presented here demonstrates that several adhesion molecules previously described are not induced when HUVEC are treated with MM-LDL (FIGURE 3).

EMAM can be isolated from endothelial cells treated with MM-LDL using such techniques as those described by Bodier (J.Biol.Chem., 256:1604-1607, 1981) for the isolation of integral membrane proteins. EMAM can be isolated by polyacrylamide gel electrophoresis (PAGE) for example, by comparing membrane prepa~ations from MM-LDL induced endothelial cells to endothelial cells which have not been exposed to MM-LDL and selecting the appropriate band from the gel. When human endothelial cells are processed in this manner, EMAM has been found to have a molecular weight of approximately 100 kD. Alternatively, for using the Western blot technique, EMAM can be identified and isolated from PAGE by using blocking antibodies raised against substantially purified EMAM andactivated cells. Furthermore, specific polyclonal or monoclonal antibodies can be used to affinity purify EMAM from MM-LDL induced endothelial cell preparations.
Techniques for purification of membrane proteins are well known to those of skill in the art and can be utilized without resorting to undue experimentation.

Like VCAM-1, ICAM-1, and ELAM-1, EMAM appears to be a glycoprotein. In the rabbit, 35S methionine labeling demonstrated three induced bands (90, 70, and 40kD). 1251 surface labeling confirmed the induction of the 90 and 70 kD bands andalso demonstrated MM-LDL induction of two larger bands at 140 and 180 kD.
Reduction of apparent molecular weight by glycopeptidase treatment and blocking of increased binding by tunicamycin suggest that the molecule is a glycoprotein.

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DiCorleto, et al. (J.lmmunol., 143:3666-3672, 1989) hypothesized that specific cell surface carbohydrate groups are required for the adhesion of monocytes to the endothelium pretreated with IL-1. Several sugars inhibited increased monocyte binding by MM-LDL suggesting that sugars may be important in binding. Since the sugars had to be present during binding to block monocyte binding, the active sugar moiety is likely on the monocyte.

Protein kinase C has been considered to be a potential pathway of endothelial cell activation by LPS, TNF, and IL-1 (Magnuson, et al., Surgely, 106:216-223, 1989).It has been shown with the present invention, that H7, which blocks PKC, effectively inhibited the induction of monocyte adherence to endothelial cells following treatment with MM-LDL.

The inventors have demonstrated that exposure of HAEC and smooth muscle cells to MM-LDL produced 2 to 3 fold more chemotactic activity (Cushing, et al., Proc.Natl.Acad.Sci. USA, 87:513~5138, 1990). This increased chemotactic activitywas shown to be due entirely to MCP-1. However, as shown here, inhibiting the activity of MCP-1 did not inhibit the increased monocyte adherence induced by MM-LDL, suggesting that this chemotactic factor is not involved in the inductionof EMAM.

The fibronectin (FN) effect on monocyte binding to endothelium cells treated with MM-LDL was tested because human endothelial cells are grown on FN coated dishes and there is some evidence that at much higher concentrations of oxidizedLDL than those employed in these studies, or with LDL which has been extensively oxidized, there may be cytotoxicity to other cell types (Chisolm, et al., Aneriosclerosis, 1:359, 1981), leading to cell detachment or disruption of cell-cell ~- 25 contacts. This might result in FN exposure and monocyte binding to FN.
Consequently, RAEC and HUVEC were tested for the effect of FN on the monocyte binding. These studies showed that anti-FN did not inhibit monocyte binding induced by MM-LDL. In addition, no increase in surface FN induced by ;~03~345 11. PATENT
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MM-LDL was observed by RIA. Microscopic evaluation showed only intact monolayers with no evidence of toxicity.

These studies show that MM-LDL induces a previously unidentified molecule on endothelial cells which is specific for monocyte binding and which may be important in the recruitment of monocytes into the early lesion of diseases suchas atherosclerosis. This novel receptor has been given the same EMAM
(Endothelial Cell Monocyte Adhesion Molecule).

The term "substantially pure" or "substantially purified" is meant to denote that the protein is substantially free of other compounds with which it is normally associated. The term is meant to describe a protein which is homogeneous by one or more purity or homogeneity characteristics used by those of ordinary skill in the art.

The term 'Yragmenr' is meant to include both synthetic and naturally-occurring amino acid or sugar sequences derivable from the naturally-occurring sequence.

The discovery of EMAM makes possible the diagnosis and therapy of EMAM-mediated pathologies. For example, EMAM can be used to produce polyclonal or monoclonal antibody preparations which are specifically reactive with this receptor. These antibodies may then be used both in vitro and in vivo for diagnosis and therapy. The term ~EMAM-agents" as used herein is meant to include such antibodies as well as EMAM receptors, EMAM ligand, inhibitors of the EMAM receptor, and fragments of these molecules. The EMAM agents, in turn, will be specific for either the EMAM receptor or EMAM ligand. For example,the diagnosis or therapy of the EMAM receptor could utilize antibodies specific for the EMAM receptor, EMAM ligand, inhibitors of the EMAM receptor, or fragments of these molecules; whereas, the diagnosis or therapy of the EMAM ligand could utilize antibodies specific for ths EMAM ligand, EMAM receptor, or fragments of these molecules.

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The gen~ral method for the production of hybridomas secreting monoclonal antibodies is well known (Kohler, et al., European J.lmm., 6:292, 1976). The isolation of hybridomas secreting monoclonal antibodies specifically reactive with EMAM can be accomplished using routine screening techniques, such as by determining if a given monoclonal antibody binds to substantially purified EMAM,or binds to endothelial cells induced to express EMAM, but do not bind to endothelial cells which do not express EMAM.

The term "antibody" as used in this invention is meant to include intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding an epitopic determinant.

The EMAM-specific antibodies, EMAM ligand, and EMAM receptor inhibitors of the invention are useful in assays in which they can be utilized in liquid phase or bound to a solid phase carrier to detect the presence of EMAM receptor in a sample. In addition, the inventive molecules used in these assays can be detectably labeled in various ways. Examples of types of assays which can be utilized to detect EMAM receptor are competitive and non-competitive assays in either a direct or inditect format. Such assays include the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the EMAM receptor can be done utilizing assays which are run in either the forward, teverse, or simultaneous modes, including histochemical assays on physiological samples.
Similarly, EMAM ligand specific agents can be utilized in such assay formats to detect EMAM ligand.

While the in vivo use of a monoclonal antibody from a foreign donor species in a different host recipient species is usually uncomplicated, a potential problemwhich may arise is the appearance of an adverse immunological response by the host to antigenic determinants present on the donor antibody. In some instances,this adverse response can be so severe as to curtail the in vivo use of the donor X03~73~S
1 3. PATENT

antibody in the host. Further, the adverse host response may serve to hinder theefficacy of the donor antibody. One way in which it is possible to circumvent the likelihood of an adverse immune response occurring in the host is by using chimeric antibodies (Sun, et al., Hybridoma, 5 (Supplement 1):S17, 1986; Oi, et al., Bio Techniques, g-o~:214, 1986). Chimeric antibodies are antibodies in which thevarious domains of the antibodies' heavy and light chains are coded for by DNA
from more than one species. Typically, a chimeric antibody will comprise the variable domains of the heavy (VH) and light (V~) chains derived from the donor species producing the antibody of desired antigenic specificity, and the variable domains of the heavy (CH) and light (CL) chains derived from the host recipient species. It is believed that by reducing the exposure of the host immune system to the antigenic determinants of the donor antibody domains, especially those inthe CH region, the possibility of an adverse immunological response occurring inthe recipient species will be reduced.

Under certain circumstances, monoclonal antibodies of one isotype might be more preferable than those of another in terms of their diagnostic or therapeutic efficacy. For example, from studies on antibody-mediated cytolysis, it is known that unmodified mouse monoclonal antibodies of isotype gamma-2a and gamma-3 are generally more effective in Iysing target cells than are antibodies of the gamma-1 isotype. This differential efficacy is thought to be due to the ability of the gamma-2a and gamma-3 isotypes to more actively participate in the cytolytic destruction of target cells. Particular isotypes of a monoclonal antibody can beprepared either directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proceedings of the Nationa/ Academy of Science, U.S.A., 82:8653, 1985; Spira, et a/., Journal of Immunological Methods, 74:307, 1 984).

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When the monoclonal antibodies of the invention are used in the form of fragments, such as, for example, Fab and F(ab')2, and especially when these fragments are therapeutically labeled, any isotype can be used since amelioration of the EMAM mediated pathology in these situations is not dependent upon complement-mediated cytolytic destruction of those cells bearing the EMAM
receptor or ligand.

The term "EMAM-mediated pathology" denotes disorders in which the EMAM
receptor contributes to the disease condition either directly or indirectly and includes cells of non-endothelial origin which have the EMAM receptor on their surface. Examples of disorders which are mediated by the EMAM receptor includes atherosclerosis, autoimmune disease, and malignancy. Malignancies of particular relevance are lipid related tumors, such as colon carcinoma and breast cancer.

The EMAM agents of the invention can be bound to many different carriers and used to detect the presence of EMAM receptor or EMAM ligand. Examples of well-known carriers includa glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can b0 either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding the EMAM agents, or will be able to ascertain such, using routine experimentation.

There are many diflerent labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the EMAM

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agent, or will be able to ascertain such, using routine experimentation.
Furthermore, the binding of these labels to the EMAM agent of the invention can be done using standard techniques common to those of ordinary skill in the art.

For purposes of the invention, EMAM receptor or EMAM ligand can be detected by the EMAM agents of the invention when present in biological fluids and tissues.
Any sample containing a detectable amount of EMAM receptor or ligand can be used. Normally, a sample is a liquid such as urine, saliva, cerebrospinal fluid,blood, serum and the like, or a solid or semi-solid such as tissues, feces, and the like.

In using the EMAM agents of the invention for the in vivo detection of EMAM
receptor or ligand, the detectably labeled agent is given in a dose which is diagnostically eflective. The term ~diagnostically effective" means that the amount of detectably labeled agent is administered in sufficient quantity to enable detection of the site having EMAM receptor or ligand for which the agent is 1 5 speciflc.

The concentration of detectably labeled agent which is administered in vivo should be sufficient such that the binding to those cells having the EMAM receptor or ligand is detectable compared to the background signal. Further, it is desirablethat the detectably labeled agent be rapidly cleared from the circulatory systemin order to give the best target-to-background signal ratio.

As a rule, the dosage of detectably labeled agent for in vivo diagnosis will vary depending on such factors as age, sex and extent of disease of the individual The dosage of agent can vary from about 0.01 mg/m2 to about 20 mg/m2 preferably about 0.1 mg/m2 to about lOmg/m2.

For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting a given radioisotope. The radioisotope chosen must .

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have a type of decay which is detectable for a given type of instrument. Stiil another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized. Ideally, a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to the EMAM agent either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to EMAM agents are bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.

The EMAM agents of the invention can also be labeled with a paramagnetic isotope for purposes of in YiVo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any sonventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI.

The EMAM agents the invention can also be used to monitor the course of amelioration of an EMAM-mediated pathology in an individual. Thus, by measuring the increase or decrease in the number of cells with EMAM receptor or ligand, or changes in the concentration of EMAM receptor or ligand shed into various body ~luids, it is possible to determine whether a particular therapeutic regimen aimed at ameliorating the EMAM mediated pathology is effective.

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The term "ameliorate" denotes a lessening of the detrimental affect of the EMAM
mediated pathology in the animal receiving therapy. The term "therapeutically effective" means that the amount of EMAM agent used is of sufficient quantity toameliorate the cause of disease due to the mediation of cells expressing EMAM
receptor or ligand. The term "animal" includes both humans and non-humans.

The EMAM agents of the invention can also be used for immunotherapy in an animal having an EMAM mediated pathology. When used in this manner, the dosage of EMAM agent can vary from about 10 mg/m2 to about 2000 mg/m2.

When used for in vivo therapy, the EMAM agents of the invention may be unlabeled or labeled with a therapeutic molecule. These molecules can be coupled either directly or indirectly to the EMAM agents of the invention. One example of indirect coupling is by use of a spacer moiety. These spacer moieties, in turn, can be either insoluble or soluble (Diener, et al., Science, 231:148, 1986) and can be selected to enable drug release from the EMAM agent at the target site. Examples of therapeutic molecules which can be coupied to the EMAM
agents of the invention for therapy are drugs, radioisotopes, lectins, and toxins.
The drugs with which can be conjugated to the EMAM agents of the invention include compounds which are classically referred to as drugs such as for example, mitomycin C, daunorubicin, and vinblastine.

In using radioisotopically conjugated EMAM agent of the invention for therapy certain isotopes may be more preferable than others depending-on such factors as target cell dlstribution as well as isotope stability and emission. If desired, the target cell distribution can be evaluated by the in vivo diagnostic techniques described above. Depending on the EMAM mediated pathology some emitters may be preferable to others. In general, alpha and beta particle-emitting radioisotopes are preferred. For solid malignancies, short range, high energy alpha emitters such as 212Bi are preferred. Examples of radioisotopes which can X03~ 45 1 8. PATENT

be bound to the antibodies of the invention for therapeutic purposes are 1251, 1311, 90~, 67CU 212Bj 211At 212pb, 47Sc, 109Pd, and 1~8Re.

Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate Iymphocytes. However, ricin is a toxic lectin which has been used therapeutically.
This is accomplished by binding the alpha-peptide chain of ricin, which is responsible for toxicity, to the EMAM agent to enable site specific delivery of the toxic effect.

Toxins are poisonous substances produced by plants, animals, or microorganisms that, in sufficient dose, are often lethal. Diphtheria toxin is a substance produced by Corynebacterium diphtheria which can be used therapeutically. This tcxin consists of an alpha and beta subunit which under proper conditions can be separated. The toxic A component can be bound to an EMAM agent and used for site specific delivery to a cell expressing EMAM receptor or ligand for which the EMAM agent of the invention is specific. Other therapeutic molecules which can be coupled to the EMAM agents of the invention are known, or can be easily ascertained, by those of ordinary ski!l in the art.

The dosage ranges for the administration of the EMAM agents of the invention arethose large enough to produce the desired effect in which the symptoms of the EMAM mediated pathology are ameliorated. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary from abou~ 0.1 mg/m2 to about 2000 mg/m2, preferably about 0.1 mg/m2 to about 500 mg/m2/dose, in one or more dose administrations daily, for one or several days. Generally, when the antibodies of the invention are administered conjugated with therapeutic Z0373~
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molecules, lower dosages, as compared to those used for in vivo immuno-diagnostic imaging, can be used.

The EMAM agents of the invention can be administered parenterally by injection or by gradual perfusion over time. The EMAM agents of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The EMAM agents of the invention can be utilized as therapeutic agents when incorporated in a solid phase matrix. The matrix can then be implanted in an animal having an EMAM-mediated disorder to allow gradual in vivo release of the agent over time. In traditional drug delivery, it has long been.recognized that tablets, capsules, and injections may not be the best mode of administration.
These conventional routes often involve frequent and repeated doses, resulting in a "peak and valleyl' pattern of therapeutic concentration. Since each therapeutic has a therapeutic range above which it is toxic and below which it is ineffective, a fluctuating therapeutic concentration may cause alternating periods of ineffectiveness and toxicity. For this reason, controlled releasa provides a way of maintaining the therapeutic agent level within the desired therapeutic range for the 20. PATENT

duration of treatment. Using a polymeric carrier is one effective means to deliver the therapeutic locally and in a controlled fashion (Langer, et a/., Rev.Macro.Chem.Phys., C23(1J, 61, 1983). As a result of less total drug required, systemic side effects can be minimized.

Polymers have been used as carriers of therapeutic to effect a localized and sustained release (Controlled Drug Delivery, Vol. I and ll, Bruck, S.D., (ed.), CRC
Press, Boca Raton, FL, 1983; Novel Drug Delivery Systems, Chien, Y.W., Marcel Dekker, New York, 1982). These therapeutic delivery systems simulate infusion and offer the potential of enhanced therapeutic efficacy and reduced systemic 1 0 toxicity.

For a non-biodegradable matrix, the steps leading to release of the therapeutic are water diffusion into the matrix, dissolution of the therapeutic, and out-diffusion of the therapeutic through the channels of the matrix. As a consequence, the mean residence time of the therapeutic existing in the soluble state is longer for a non-biodegradable matrix than for a biodegradable matrix where a long passage through the channels is no longer required. Since many pharmaceuticals have short half-lives it is likely that the therapeutic is decomposed or inactivated inside the non-biodegradable matrix before it can be released. This issue is particularly significant for many bio-macromolecules and smaller polypeptides, since these molecules are generally unstable in buffer and have low permeability through polymers. In fact, in a non-biodegradable matrix, many bio-macromolecules will aggregate and precipitate, clogging the channels necessary for diffusion out of the carrier matrix. This problem is largely alleviated by using a biodegradable matrix which allows controlled release of the therapeutic.

Biodegradable polymers differ trom non-biodegradable polymers in that they are consumed or biodegraded during therapy. This usually involves breakdown of the polymer to its monomeric subunits, which should be biocompatible with the surrounding tissue. The life of a biodegradable polymer in vivo depends on its ~03734~
21. PATENT

molecular weight and degree of cross linking; the greater the molecular weight and degree of cross linking, the longer the life. The most highly investigated biodegradable polymers are polylactic acid (PIA), polyglycolic acid (PGA), copolymers of PLA and PGA, polyamides, and copolymers of polyamides and polyesters. PLA, sometimes referred to as polylactide, undergoes hydrolytic deesterification to lactic acid, a normal product of muscle metabolism. PGA is chemically related to PLA and is commonly used for absorbable surgical sutures, as is PWPGA copolymer.

An advantage of a biodegradable material is the elimination of the need for surgical removal after it has fulfilled its mission. The appeal of sueh a material is more than simply for convenience. From a technical standpoint, a material which biodegrades gradually and is excreted over time can offer many unique advantages.

A biodegradable delivery system has several additional advantages: 1 ) the therapeutic release rate is amenable to control through variation of the matrix composition; 2) implantation can be done at sites difficult or impossible for retrieval; 3) delivery of unstable therapeutic is more practical. This last point is of particular importance for polypeptides where short in vivo half-lives and low Gltract absorption often render them unsuitable for conventional oral or intravenous administration. Also, because these substances are often unstable in buffer, such polypeptides cannot be effectively delivered by pumping devices.

In its simplest form, an EMAM agent delivery system consists of a dispersion of the agent in a polymer matrix. The agent is released as the polymeric matrix decomposes, or biodegrades into soluble products which are excreted from the body. Several classes of synthetic polymers, including polyesters (Pitt, et al., in Controlled ~qelease of Bioactive Materials, R. Baker, Ed., Academic Press, New York, 1980); polyamides (Sidman, et al., Jouma/ of Memorane Science, _:227, 1979); polyurethanes (Maser, et al., Joumal of Polymer Science, Polymer ;~03~34~
22. PATENT

Symposium, 66:259, 1979); polyorthoesters (Heller, et al., Polymer Engineering Science, 21:727, 1981); and polyanhydrides (Leong, et al, 13iomaterials, 7:364, 1986) have been studied for this purpose.

By far most research has been done on the polyesters of PLA and PLA/PGA.
Undoubtedly, this is a consequence of convenience and safety considerations.
These polymers are readily available, as they have been used as biodegradable sutures, and they decompose into non-toxic lactic and glycolic acids.

Polyorthoesters and polyanhydrides have been specifically designed for controlled release purposes. By taking advantage of the pH dependence of the rate of orthoester cleavage, preferential hydrolysis at the surface is achieved by either the addition of basic substances to suppress degradation in bulk, or the incorporation of acidic catalysts to promote surface degradation.

The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the antibodies of the invention, the medicament being used for therapy of EMAM-mediated pathologies.

As described above, the diagnosis and therapy of EMAM mediated pathologies can also be achieved using EMAM receptor, EMAM ligand, inhibitors of the EMAM
receptor, or fragments of these molecules. EMAM peptides can be used to bind to the EMAM ligand on monocytes to block the ability of a monocyte to bind to an endothelial cell. EMAM ligand or carbohydrate inhibitors could ba utilized similarly but would block the adhesion of monocytes to endothelial cells by binding to the EMAM receptor. When EMAM receptor is utilized, it is preferable to provide a molecule which is devoid of the trans membrane region such that solubility is enhanced (Fisher, et al., Nature, 331:76, 1988). The use of EMAM
receptor, EMAM ligand, and carbohydrate inhibitors is preferred to the use of antibody therapy where the probability of an adverse immune response is more likely to occur.

~03~345 23. PATENT

The presence of EMAM on other cell types, such as tumor cells, enhances the value of using EMAM receptor, EMAM ligand, or inhibitory carbohydrates diagnostically and therapeutically. From a therapeutic standpoint, in addiUon tothe therapeuUc modes described above, where the EMAM mediated pathology is a tumor which expresses the EMAM receptor, the present invention allows the utilization of more advanced therapeutic methodologies such as tumor inhltratingIymphocyte (TIL) immunotherapies. Thus, where a malignancy expresses EMAM
receptor, the tumor can be biopsied and Iymphocytes which have infiltrated the tumor removed and isolated. A retroviral expression vector carrying a gene for a tumoricidal agent, such as tumor necrosis factor (TNF), is used to then transfect the TIL The TIL are then expanded using IL-2 and injected into the patient wherethey migrate back to the tumor from which they were derived whereupon the tumoricidal gene is expressed and released to react with the tumor cells.

The ability to induce EMAM receptor using MM-I DL provides a convenient model for screening molecules which may be used to inhibit the adhesion phenomenon between monocytes and endothelial cells expressing EMAM receptor. In tne present invention, studies using carbohydrates appear to suggest that molecules with the structure:
c~,,o ~
~ >~ o R
o X

where R is hydrogen or phosphonyl; X is hydroxyl, amino, or protected amino;
and Y is hydrogen or a saccharide residue, are particularly effective in inhibiting the adhesion phenomenon.

;~03~5 24. PATENT

The knowledge gained from the activities of these small molecular inhibitors allows for the identification of endogenous in vivo molecules which can also inhibit monocyte binding to endothelial cells expressing the EMAM receptor. Also, the EMAM receptor of fragments thereof can be used as an affinity reagent for the purification of the EMAM ligand. If desired, the EMAM receptor can be immobilized to a solid phase as described above. (\Nheeler, et al., J. Clin.lnvest, 82:1211, 1988).

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

REGULATION OF BINDING

Various cell populations were prepared using standard techniques. RAEC at passages 6-17 and HUVEC at passages 1-4 were prepared as described by Barliner, et al. ~4teriosclerosis, 6:254, 1986). Monocytes were isolated by a modification of the method of Recalde (Fogelman, et al., J.Lipid Res. 198, 29:1243 1247, 1988). The human monocyte cell lines THP-1 (ATCC TIB 202) and U937 (ATCC CRL 1593) were also used as a source of monocyte-like cells.
Neutrophils and Iymphocytes were prepared from human plasma employing previously described methods (A. Boyum, Scand. J. Clin., 21:77-89, 1968; Van Epps, et al., J.lmmunol., 143:3207-3210, 1989). HL 60 (ATCC CCL 240) was also used as a source of neutrophil-like cells.

MM-LDL was prepared starting with low density lipoprotein (LDL). LDL was isolated by density gradient centrifugation of serum and stored in phosphate buffered 0.15 M NaCI containing 0.01% EDTA. MM-LDL was obtained by storage X037;~5 25. PATENT

of LDL at 4OC for 3-6 months or by mild iron oxidation. These minimally oxidizedlipoproteins contain 2-5 nanomoles thiobarbituric acid reactive substances [TBARS]/mg cholesterol. TBARS include malondialdehyde and other fatty acid oxidation products such as alkenes and hydroperoxides. (A. Boyum, Scand. J.
Clin., 21:77-89, 1968). The time of iron oxidation to produce active LDL varied considerably with the LDL preparation and the presence of a particular amount ofTBARS in the preparation did not guarantee activity. Therefore, times of exposure to iron from 4 hours to 72 hours were routinely employed and preparations oxidized for various times screened for activity. It was observed that cells exhibit a varying susceptibility to MM-LDL. Consequently, wherever possible, cells from particular individuals used for screening, were used for experiments.
Approximately 1/2 of the LDL preparation prepared by iron oxidation was found to result in material active on P~EC. Although it was more difficult to obtain LDL
active on HUVEC, about 1/4 of the preparations were active.

For studies on cellular adhesion, P~EC and HUVEC were rinsed with serum free media and 2 x 105THP-1,or monocytes in RPMI containing 1% FCS were added to each well. After a 45 minute incubation, the nonadherent cells were removed by washing and the wells were fixed with 1% glutaraldehyde. The number of attached cells was determined visually or in some cases, by measuring radioactivity of 3H thymidine or 5~Cr labeled THP-1 adherent to the monolayer (Pawlowski, et al., Proc.Natl.Acad.Sci. USA, 82:8208-8212, 1985). In the case ofU937 and T-cell adhesion to HUVEC, cells suspended in PBS were injected into adhesion chambers and allowed to interact with the endothelial monolayer for 10 min. The chambers were then inverted for 500 seconds to remove unattached cells (Smith, etal., J. Clin.lnvest., _:2008-2017,1989).

Earlier studies indicated that P~EC treated for 4 hours with 1-5 ~g/ml of MM-LDL(obtained by storage technique) were induced to bind monocytes, but not neutrophils. To compare the binding molecule to known adhesion molecules, human cells had to be employed.

~3734S

26. PATENT

Four hour pretreatment of HUVEC with MM-LDL at 100 ~g/ml induced a 47-fold increase in the adhesion of THP-1 to endothelial cells. This increase was similar to that seen with IL-1 pretreatment (10 U/ml). HL60 adhesion was not signiflcantly stimulated by MM-LDL, but was stimulated by IL-1 (FIGURE 1). In a separate experiment, the effect of MM-LDL on the binding of Iymphocytes to HUVEC was examined. Pretreatment of HUVEC with MM-LDL (100 ~g/ml for 4 hours) induced binding of the U937 cells, a monocytic cell line, but did not induce the Iymphocyte binding to the endothelial cells; however, Iymphocyte binding was increased by 4-fold with LPS (10 ng/ml) pretreatment (FIGURE 2). It is important to note thatthe higher concentrations of MM-LDL were used for these studies as opposed to previous studies. Because HUVEC are less sensitive than FtAEC to the effects of MM-LDL, concentrations up to 500 ~lg/ml may be necessary for effects.

IMIAUNOCHEMICAL CHARACTERIZATION

Radioimmunoassays and ELISA were used to characterize the monocyte adhesion molecule on endothelial cells induced with MM-LDL. These immunoassays utilized antibodies to several known adhesion molecules after treating HUVEC with MM-LDL (100 ~.g/ml), and IL-1 (10 U/ml).

HUVEC in 96-well dishes were pretreated with medium containirlg MM-LDL (100 ~.g/ml), IL-1 (10 U/ml), or no additives for 4 hours. The wells were rinsed twice with ice cold PBS containing calcium, magnesium, glucose, and 5% Fetal bovine serum. Cells were treated with monoclonal antibodies (ELAM-1 :P6E2, VCAM-1:p1B5, ICAM-1:P3G1) for 1 hour, and then ~251-labeled or peroxidase labeled, goat anti-mouse second antibodies were added for 2 hours. All antibodies were obtained from Cytel Corp. (San Diego, CA). P6E2, an IgG3, ;~03~ 5 27. PATENT

crossreacted with the ELAM-1 protein immunoprecipitated with H1 87 (Bevilacqua, etal., Proc.Natl.Acad.Sci.USA, 84:9238-9242, 1987); PlB5, an IgG1, crossreacted with VCAM-1 protein immunoprecipitated by antibody 489 (Carlos, et al., Blood, 76:965-970, 1990); and P3G1, an IgG1, crossreacted with ICAM01 protein immunoprecipitated by RR1.1 obtained from Boehringer Mannheim. Unbound antibodies were removed by washing. The cells were dissolved in 2hl NaOH and radioactivity determined, or o-phenylenediamine-peroxide was added and absorbance was read at 492 nm on a Titertek Multiscan MCC/340.

These studies showed that IL-1 induced ELAM-1, VCAM-1, and ICAM-1, howel/er, none of these molecules was significantly induced by MM-LDL (FIGURE 3). In fact, in this study, the amount of ICAM-1 was decreased while in a separate study employing another monoclonal antibody (R 6.5), ICAM-1 remained unchanged.
This lack of induction of ICAM-1 and ELAM-1 was shown on three different MM-LDL preparations where binding was simultaneously measured. Lack of 1 5 VCAM-1 induction was testad simultaneously with binding on only the preparation shown, but was also shown to be negative in a second preparation where THP-1 binding was assayed separately. Treatment of monocytes with anti CD 1 8 antibody (TS1 /1 8) had little effect on MM-LDL induced monocyte binding suggesting that this family of integrins is also not involved in binding.

In other experiments, endothelial cells were pretreated with the Fab fragments of polyclonal antibodies to fibronectin or MCP-1 and then tested for monocyte binding. Fab fragments of either polyclonal antibody to fibronectin (Calbiochem,San Diego, CA) or to Monocyte chemotactic peptide (MCP-1) (obtainecl from Anthony J. Valente, The Cleveland Research Institute, Cleveland, OH) were prepared according to previous methods (Ishikawa, et al., J. Immunol.Me~hods, 38: 11 7-1 23, 1 980). Endothelial cells were treated with 10 ~g/ml of Fab fragments for 30 minutes. Monocytes were then added to endothelial monolayers in the presence or absence of antibody. For studies of antibody effects on monocytes, the monocytes were pretreated with 10 ~.g/ml of antibody (TS1/18, provided from .

203~7345 28. PATENT

C. Wayne Smith, 8aylor College of Medicine, Houston TX) for 30 minutes, then monocytes were added to MM-LDL treated or untreated cells.

In these experiments, none of these antibodies blocked the increased binding induced by MM-L~L (FIGURE 4). In addition, it was shown by ELISA that FN
production was not inducsd by MM-LDL.

BIOCHEMICAL CHARACTERIZATION

Treating RAEC for 2 hours with cycloheximide (1 ~Ig/ml) and tunicamycin (4 ~g/ml) prior to the addition of the MM-LDL caused a 60% reduction in monocyte binding (FIGURE 5). Under the conditions employed, cycioheximide inhibited protein synthesis by 80% while tunicamycin had no effect on total protein synthesis but inhibited incorporation of 3H glycosamine into protein by 35-60%, suggesting that the binding molecule was a glycoprotein. The effect of several sugars on monocyte adhesion was tested.

After treatment of RAEC cells with MM-LDL for 4 hours, cells were rinsed with ice cold PBS, and fixed with 1% paraformaldehyde in 1M sodium cacodate for 20 minutes at 4OC. To measure the effect of sugars, fixed cells were washed two times with ice cold PBS and incubated with sugars; lactose-1-phosphate (10~M), maltose-1-phosphate (10~M), N-acetyl-glycosamine (102M), fructose-~phosphate (102M), mannose-6-phosphate (10-2M), and glucose-6-phosphate (10-2M) for 30 minutes at 4~C or 37C before adding THP-1 cells. The adhesion assay was carried out in the presence or absence of the sugars. 0f the tested sugars, onlylactose-1 -phosphate, maltose-1-phosphate, and N-acetyl-glycosamine blocked the binding by 90-100% (FIGURE 6). Other sugars including mannose-6-phosphate, fructose-6-phosphate, and glucose-6-phosphate did not inhibit binding.

~03734~5 29. PATENT

To determine whether calcium or magnesium are necessary for monocyte binding, THP-1 cells rinsed twice with 1% PBS and resuspended in medium with 1 mM
EDTA or EGTA (Stoolman, et al., Blood, 70:1842-1850, 1987). Tnese THP-1 cells were added to the fixed endothelial cells with or without 3 or 6 mM of calcium or magnesium and the number of adherent cells determined as described above.
EDTA and EGTA treatment reduced binding by more than 90% and this was reversed by the addition of calcium or magnesium (FIGURE 7).

The effect of H7 and HA1004 (two isoquinoline-sulfonamide derivatives that inhibit protein kinases by competing for the ATP-binding site but differ with respect toprotein inase C[PKC] in that H7 effectively inhibits PKC and HA1û04 does not) oninduction of binding was examined. H7 inhibited binding by 85% while HA1004 was without effect (FIGURE 8) suggesting that PKC is involved in the MM-LDL
induction of the binding protein.

To estimate the molecular weight of EMAM, autoradiography of SDS
polyacrylamide gels was used to analyze the 35S methionine labeled plasma membrane proteins.

Integral membrane proteins were isolated by the method of Bodier (J. Biol.Chem, 256:1604-1607, 1981) using Triton X-114. 8riefly, F~EC or HUVEC were treated with MM-LDL or Lipopolysaccharide (LPS, 1 ~g/ml, from E. coli strain 0111 :B4, Ust Biological Laboratories) for 4 hours. To label the cells with 35S methionine, they were incubated with 500 uCi 35S methionine in methionine free media during the final 30-45 minutes of MM-LDL treatment. Cells were then Iysed in 1% Triton X-114 in 10 mM Tris-HCI, 150 mM NaCI, PH 7.4, 1mM PMSF, 20 ~g/ml pepstatin and leupeptin. The Iysate was centrifuged at 12,000 9 for 5 minutes at 4C. The supernatant was layered on a sucrose cushion, warmed to 37~C for 5 minutes, and centrifuged for 3 minutes at 300 9 at room temperature. The detergent phase was subjected to trichloroacetic acid precipitation and was analyzed by SDS
polyacrylamide gel electrophoresis (Laemmli, U.K., Nature [Lond.~, 227:680-, 1970).

X03~3~
30. PATENT

In some cases, the isolated 35S methionine labeled membrane proteins were incubated with ~Iycopeptidase F (2 U/ml) in 0.2 M NaPO4, PH 8.6,1.25% NP-40, and 0.01 M ~-mercaptoethanol for 18 hours. The gels were fixed, dried, and subjected to autoradiography on X-ray film.

Equal amounts of TCA precipitable radioactivity from treated and untreated cellswere subjected to electrophGresis under reducing conditions. Increased incorporation of 35S methionine in the MM-LDL treated cells was seen in three bands of approximately 90,000, 70,000, and 40,000 M.W. (FIGURE 9). This result was reproducible for different strains and passages of RAEC. In human aortic endothelial cells, induction of only a 100,000 M.W. band was observed after MM-LDL treatment (FIGURE 11). FIGURE 10 shows the sarne 35B methionine labeled membrane preparation after treatment with glycopeptidase.
Glycopeptidase treatment reduced the molecular weight of two of the induced bands, 90,000 to 75,000 M.W. and 40,000 to 35,000 M.W., suggesting that these molecules are highly glycosylated.

Radioiodination was carried out as described by Hubbard and Cohn (J. Cell Biol.,64:438-460, 1975). Cells were incubated in an ice water bath for 30 minutes, in a PBS containing 100-300 uCi/ml carrier-free Na1251, 20 mM glucose, and 8-12 uU
glucose oxidase. Iodination was stopped by aspiration of the solution, washed serially with 0.02% NaN3 in PBS, followed by 0.1 M Nal in PBS, and then PBS.
The cells were Iysed with 1% Triton X-114 solution and processed as described above.

Radioiodination of cell surface protein molecules also demonstrated an increase in radioactive bands at 90,000 and 70,000 M.W., but not the 40,000 M.W. band.
In addition to these, 140,000 and 180,000 M.W. bands were seen in MM-LDL
treated samples.

31. PATENT

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made without departing from the spirit or scope of the invention.

Claims (54)

1. Substantially pure EMAM or a fragment thereof.

2. A process useful in inducing the expression of EMAM which comprises contacting an endothelial cell with MM-LDL.

3. The process of claim 2, wherein the lipoprotein is oxidized by storage under physiologic conditions at about 4°C for a period of from about 2 months to about 8 months.

4. The process of claim 2, wherein the lipoprotein is oxidized using a chemical.

5. The process of claim 4, wherein the chemical is an iron salt.

6. The process of claim 5, wherein the salt is ferrous sulfate.

7. A process of making MM-LDL capable of inducing expression of EMAM
which comprises:

a) isolating LDL;
b) oxidizing the isolated LDL to produce MM-LDL;
c) screening the activity of the MM-LDL by exposing an endothelial cell to the MM-LDL; and d) determining the presence of EMAM on the surface of the endothelial cell.

8. The process of claim 7, wherein the LDL is oxidized by storage under physiologic conditions at about 4°C for a period of from about 2 months to about 8 months.

33. PATENT

9. The process of claim 7, wherein the LDL is oxidized using a chemical.

10. The process of claim 9, wherein the chemical is an iron salt.

11. The process of claim 10, wherein the salt is ferrous sulfate.

12. The process of claim 9, wherein the oxidizing chemical contains from about 1 to about 10mM of TBARS/mg cholesterol.

13. A method of identifying molecules which inhibit binding of EMAM ligand to EMAM receptor comprising:

a) creating a first mixture by contacting a molecule with an EMAM receptor;
b) incubating the components of step a) under conditions and for a period of time sufficient to allow the molecule to bind to the EMAM receptor;
c) creating a second mixture by contacting the first mixture with EMAM
ligand;
d) incubating the components of step c) under conditions and for a period of time sufficient to allow the EMAM receptor to bind with the EMAM
ligand; and e) measuring the degree of binding between the EMAM receptor and the EMAM ligand.

14. The method of claim 13, wherein the EMAM receptor is on an endothelial cell.

15. The method of claim 13, wherein the EMAM ligand is on a monocyte.

16. The method of claim 13, wherein the EMAM receptor is detectably labeled.

17. The method of claim 13, wherein the EMAM ligand is detectably labeled.

18. A method for purifying a member of the EMAM receptor/EMAM ligand binding pair comprising:

a) contacting a sample containing the member with a member binding agent;
b) incubating the components of step a) under conditions and for a period of time sufficient to allow the member to bind with the member binding agent to form a complex;
c) separating the complex from the remainder of the sample; and d) recovering the member from the separated complex.

19. The method of claim 18, wherein the member is EMAM receptor and the member binding agent is selected from the group consisting of an EMAM
ligand, an antibody specific for EMAM receptor, and a lectin.

20. The method of claim 19, wherein the antibody is monoclonal.

21. The method of claim 18, wherein the member is EMAM ligand and the member binding agent is selected from EMAM receptor, an antibody specific for EMAM ligand, and a lectin.

22. The method of claim 21, wherein the antibody is monoclonal.

23. An antibody or fragment thereof specific for a member of the EMAM
receptor/EMAM ligand binding pair.

24. The antibody of claim 23, wherein the antibody is polyclonal.

25. The antibody of claim 23, wherein the antibody is monoclonal.

35. PATENT

26. The antibody of claim 23, wherein the member is the EMAM receptor.

27. The antibody of claim 23, wherein the member is the EMAM ligand.

28. A method for producing antibodies specific for a member of the EMAM
receptor/EMAM ligand binding pair which comprises immunizing an animal with the member or a fragment thereof.

29. The method of claim 28, wherein the member is the EMAM receptor.

30. The method of claim 28, wherein the member is the EMAM ligand.

31. A method of ameliorating an EMAM receptor mediated pathology in an animal comprising:

administering to the animal a therapeutically effective amount of an EMAM
agent.

32. The method of claim 31, wherein the EMAM agent inhibitors adhesion between the EMAM receptor and the EMAM ligand.

33. The method of claim 31, wherein the EMAM agent is selected from the group consisting of EMAM receptor, EMAM ligand, inhibitors of the EMAM
receptor, and fragments of these molecules.

36. PATENT

34. The method of claim 33, wherein the inhibitors has the structure:

wherein R is hydrogen or phosphonyl; X is hydroxyl, amino, or protected amino; and Y is hydrogen or a saccharide residue.

35. The method of claim 33, wherein the inhibitors are selected from the group consisting of lactose-1-phosphate, maltose-1-phosphate, and N-acetyl-glucosamine.

36. The method of claim 31, wherein the EMAM agent is therapeutically labeled.

37. The method of claim 36, wherein the therapeutic label is selected from the group consisting of a radioisotope, a drug, an immunomodulator, a biological response modifier, a lectin and a toxin.

38. The method of claim 31, wherein the pathology is selected from the group consisting of atherosclerosis, inflammatory disease, autoimmune disease, and malignancy.

39. The method of claim 38, wherein the malignancy is a lipid related tumor.

40. The method of claim 39, wherein the tumor is colon carcinoma or breast cancer.

41. The method of claim 31, wherein the administration is parenteral.

37. PATENT

42. The method of claim 41, wherein the parenteral administration is by subcutaneous, intramuscular, intraperitoneal, intracavity, transdermal, or intravenous injection.

43. The method of claim 31, wherein the EMAM agent is administered in a solid phase matrix.

44. The method of claim 43, wherein the matrix is biodegradable.

45. A pharmaceutical composition comprising EMAM mediated pathology ameliorating amounts of an EMAM agent together with a pharmaceutically inert carrier.

46. A method of detecting EMAM mediated pathology which comprises contacting a source suspected of containing EMAM receptor or EMAM
ligand with a diagnostically effective amount of detectably labeled EMAM
agent and determining if the EMAM agent binds with the EMAM receptor or EMAM ligand.

47. The method of claim 46, wherein the EMAM agent inhibits adhesion between the EMAM receptor and EMAM ligand.

48. The method of claim 47, wherein the EMAM agent is selected from the group consisting of EMAM receptor, EMAM ligand, inhibitors of the EMAM
receptor, and fragments of these molecules.

38. PATENT

49. The method of claim 48, wherein the inhibitor has the structure:

wherein R is hydrogen or phosphonyl; X is hydroxyl, amino, or protected amino; and Y is hydrogen or a saccharide residue.

50. The method of claim 48, wherein the inhibitors are selected from the group consisting of lactose-1-phosphate, maltose-1-phosphate, and N-acetyl-glucosamine.

51. The method of claim 46, wherein the detecting is in vivo.

52. The method of claim 51, wherein the detectable label is a radioisotope or a paramagnetic label.

53. The method of claim 46, wherein the detecting is in vitro.

54. The method of claim 53, wherein the detectable label is selected from the group consisting of a radioisotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, and an enzyme.