WO1992014757A1 - Endothelial cell-monocyte adhesion molecule - Google Patents

Abstract

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

Description

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

This application is a continuation-in-part of U.S. Serial No. 07/660,024, filed February 25, 1991.

Field 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 cause or 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 tissue damage occurs, soluble chemical substances are elaborated which initiate the inflammatory response. The inflammatory response consists of a complex series of events which include a localized increase in blood flow, with capillary dilation and increased 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 inflammation site. This exudate initially consists primarily of polymorphonuclear leukocytes, followed by monocytes, lymphocytes, 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 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 cells have been described including: ELAM-1 , ICAM-1 , and VCAM-1. ELAM-1 has been cloned by Bevilacqua, et al., {J. Clin.lnvest, 7.6:2003-, 1985) and selectively mediates neutrophil and monocyte, but not lymphocyte, binding. ELAM-1 is induced on human umbilical vein endothelial cells (HUVEC) by IL-1 , TNF-alpha, and LPS (Pober, et al., J. Immunol., 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-I. ELAM-1 is a member of the LEC-CA family which include GMP-140 and LEC- CAM-1 (murine MEL-14 Ag, and its human homologues LA -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, §2:475-484, 1990).

A second inducible endothelial molecule, ICAM-1 , seems to be involved in all leukocyte binding (Pober, et al., J. Immunol., 127:1893-, 1986; Dustin, et al.,

J. Immunol., 127:245-254, 1986). It is induced by treatment with IL-1, TNF alpha, IFN gamma, lymphotoxin, 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 member of the immunoglobulin supergene family. LFA-1 , one of the integrin family, is a known ligand for ICAM-1. Anti-CD18 was reported to reduce monocyte adherence to unstimulated HUVEC, but had no significant effect on HUVEC pretreated with 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, et al. {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 lymphocyte 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 on the 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 be possible 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 the monocyte is an etiologic agent. The present invention addresses this need by providing a receptor with such specificity.

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 fragments, 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 likelihood of toxicity can be minimized. Another major advantage of the present invention is that it provides the art with an immunosuppressive agent which, although highly effective, can be utilized at concentrations which minimize the likelihood of host toxicity.

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 μg/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 cells were removed by washing and adherent cells were counted. Values represent mean +S.D.(n=3).

FIGURE 2: U937 and T cell adhesion to HUVEC. HUVEC monolayers were incubated with MM-LDL(40 μg/ml), or LPS (10 ng/ml) for 4 hours at 37° C, 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 10 min. 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 for LPS induced binding of U937 and T cells are significant (p<0.01) and for MM-LDL induced binding of U937. n=4 separate experiments (U937 cells), n=8 (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 ig/ml) (the same preparation was used as for Figure 1), and IL-1 (10 U/ml) for 4 hours at 37 ^β C. 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 with peroxidase labeled goat anti-mouse second antibody for 2 hours. Values are O.D. 492 from microtiter plate reader (n=5). (Values for ELAM-1 and VCAM-1 are on the y axes on the left and ICAM-1 is on the right.)

FIGURE 4: Effect of FN-Ab (fibronectin antibody) and MCP-1-Ab (monocyte chemotactic protein-1 antibodyon monocyte adhesion to HUVEC. After treatment of HUVEC with MM-LDL, endothelial monolayers were treated with 10 μg/ml of Fab fragments of MCP-1-Ab or FN-Ab, and then monocytes added in the presence (+AB) or absence of antibodies. Values represent mean +S.D. (n=4).

FIGURE 5: Effect of cycloheximide and tunicamycin on MM-LDL induced THP- 1 cell binding. RAEC were pretreated with (CH+) or without (CH-) cyclohexi¬ mide (1 μg/ml) and with (TU+) or without (TU-) tunicamycin (4 μg/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 (panel A) and HAEC (panel B) were treated with MM-LDL for 4 hours, and fixed in 1% paraformaldehye for 20 min at 4 ° C. After washing the fixed cells with PBS, the sugars lactose-1 -phosphate (L1P), maltose-1 -phosphate (M1P), N- 5 acetylglucosamine (NAG), fructose-6-phosphate (F6P), glucose-6-phosphate (G6P), mannose-6-phosphate (M6P), and glucose-1 -phosphate (G1P) were added at 10 ^M for 30 minutes. Monocyte binding as determined in the presence of sugars. Values are mean -f-S.D. (n=4).

FIGURE 7: Effect of calcium and magnesium on THP-1 binding to RAEC.

10 Before adding THP-1 cells to the fixed endothelial monolayer, THP-1 cells were rinsed with PBS (1% FCS) containing 1mM of EDTA or EGTA. Monocyte binding was determined in the presence or absence of 3 mM calcium or magnesium (EDTA experiments: panel A) or 6mM of calcium or magnesium (EGTA experiments: panel B) in a separate set of experiments. Values are

15 mean +S.D. (n=5).

FIGURE 8: Effect of H7 and HA 1004 on MM-LDL action. RAEC were pretreated with H7(100 μM/ml) or HA 1004 (100 μM) for 30 minutes at 37 ° C and then incubated with MM-LDL(2 μg/ml) for 4 hours at 37 °C. The monocyte binding assay was carried out and adherent cells counted visually. Values 20 represent mean +S.D. n=5. FIGURE 9: Induction of integral membrane proteins by MM-LDL. RAEC were treated with MM-LDL for 4 hours and labeled with ^S methionine for the last 30 minutes of incubation. Cells were lysed in 1 % Triton X-114 (in 10mM Tris- HCl, 150mM NaCl, pH 7.4, 1mM PMSF, 20 μg/ml pepstatin and leupeptin). The detergent phase was subjected to TCA precipitation, analyzed by SDS PAGE, and autoradiography. C = control and M = MM-LDL treated. Arrows indicated induced proteins.

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 37 ° C and analyzed by SDS PAGE and autoradiography. M = integral membrane proteins from MM-LDL treated cells. MG = same preparation as in M, but after glycopeptidase treatment. Arrows indicate positions of induced bands before and after glycopeptidase.

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 ^S methionine for the last 30 minutes of incubation. Cells were lysed in 1% Triton X-114 (in 10mM Tris-HCl, 150mM NaCl, pH 7.4, 1 mM PMSF, 20 μg/ml pepstatin and leupeptin). The detergent phase was subjected to TCA precipitation, analyzed by SDS PAGE, and autoradiography. C=control; M= MM-LDL treated. 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 present on monocytes which is essentially absent from neutrophils or lymphocytes.

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 exposure to UV light (Dousset et al., Biochim Biophy. Ada, 1045:219-223. 1990) or by incubation with soy bean lipoxygenase (Sparrow, et al., J. Lipid Res., 29:745-753, 1988). Oxidation conditions can be selected to produce LDL particles 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 their 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 μg/ml to about 200 μg/ml of MM-LDL. These incubation times and concentrations may vary depending on the source of the endothelial cells. For example, rabbit endothelial cells are preferably treated with from about 1 μg/ml to about 5 μg/ml of MM-LDL, whereas human endothelial cells are preferably treated from about 50 μg/ml to about 150 μg/ml of MM-LDL. Those of skill in the art can readily ascertain the 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 lymphocyte 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 preparations 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 and activated 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 ELΛM-1, EMAM appears to be a glycoprotein. In the rabbit, ^S methionine labeling demonstrated three induced bands (90, 70, and 40 kD). ²⁵l surface labeling confirmed the induction of the 90 and 70 kD bands and also 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.

DiCorleto, et al. {J. Immunol., 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., Surgery, 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, 87:5134-5138, 1990). This increased chemotactic activity was 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 induction of 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 oxidized LDL than those employed in these studies, or with LDL which has been extensively oxidized, there may be cytotoxicity to other cell types (Chisolm, ef al., Arteriosclerosis, 1:359, 1981), leading to cell detachment or disruption of cell-cell 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 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 such as 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 "fragment" 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 polyclona! 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 the EMAM ligand, EMAM receptor, or fragments of these molecules.

The general 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 indirect format. Such assays include the radioimmu- noassay (RIA) and the sandwich (immunometric) assay. Detection of the EMAM receptor can be done utilizing assays which are run in either the forward, reverse, 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 problem which 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 antibody in the host. Further, the adverse host response may serve to hinder the efficacy 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, 4(21:214, 1986).

Chimeric antibodies are antibodies in which the various 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 (V_H) and light (V_L) chains derived from the donor species producing the antibody of desired antigenic specificity, and the variable domains of the heavy (C_H) and light (C_L) 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 in the

C_H region, the possibility of an adverse immunological response occurring in the 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 lysing 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 be prepared 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 National Academy of Science, U.S.A., 22:8653, 1985; Spira, et al., Journal of Immunological Methods, 74:307, 1984).

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 amelior¬ ation 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 include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be 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 different 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 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 *n a dose which is diagnostically effective. 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 specific.

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 desirable that the detectably labeled agent be rapidly cleared from the circulatory system in 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 10mg/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 have a type of decay which is detectable for a given type of instrument. Still 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 vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional 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 fluids, it is possible to determine whether a particular therapeutic regimen aimed at ameliorating the EMAM mediated pathology is effective.

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 to ameliorate 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 coupled 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 distribution 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 be bound to the antibodies of the invention for therapeutic purposes are ¹²⁵l, ¹³¹l, ∞Y, ⁶⁷Cu, ²¹ Bi, ²¹¹At, ^{2 2}Pb, ⁴⁷Sc, ¹⁰⁹Pd, and ¹⁸⁸Re.

Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. 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 5 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 toxin consists of an alpha and beta subunit which under

10 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 skill in

15 the art.

The dosage ranges for the administration of the EMAM agents of the invention are those 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, 20 anaphylactic reactions, 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 about 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 molecules, lower dosages, as compared to those used for in vivo immunodiagnostic 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 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 valley" pattern of therapeutic concentration. Since each therapeutic has a therapeutic range above which it is toxic and 5 below which it is ineffective, a fluctuating therapeutic concentration may cause alternating periods of ineffectiveness and toxicity. For this reason, controlled release provides a way of maintaining the therapeutic agent level within the desired therapeutic range for the duration of treatment. Using a polymeric carrier is one effective means to deliver the therapeutic locally and in a con- 10 trolled fashion (Langer, et al., Rev.Macro.Chem.Phys., C23(1), 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 II, Bruck, S.D., (ed.), CRC Press, Boca Raton, FL, 1983; Novel Drug Delivery Systems, Chien, Y.W., 15 Marcel Dekker, New York, 1982). These therapeutic delivery systems simulate infusion and offer the potential of enhanced therapeutic efficacy and reduced systemic 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-diffu-

20 sion 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

25 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 con¬ trolled release of the therapeutic.

Biodegradable polymers differ from 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 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 (PLA), 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 PLA/PGA 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 such 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 5 is of particular importance for polypeptides where short in vivo half-lives and low Gl tract 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.

10 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, ef al., in Controlled Release of Bioactive Materials, R. Baker, Ed., Academic

15 Press, New York, 1980); polyamides (Sidman, ef al., Journal of Membrane Science, 7:227, 1979); polyurethanes (Maser, ef al., Journal of Polymer Science, Polymer Symposium, 66:259, 1979); polyorthoesters (Heller, ef al., Polymer Engineering Science, 21:727, 1981); and polyanhydrides (Leong, ef al., Biomaterials, 7:364, 1986) have been studied for this purpose.

20 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 be 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, ef al., Nature,

221:76, 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.

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 addition to the therapeutic 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 infiltrating lymphocyte (TIL) immunotherapies. Thus, where a malignancy expresses EMAM receptor, the tumor can be biopsied and lymphocytes 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 where they 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-LDL 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 the present invention, studies using carbohydrates appear to suggest that molecules with the structure:

where R is hydrogen or phosphono; X is hydroxyl, amino, or protected amino; and Y is hydrogen or a carbohydrate residue, with the proviso that when R is phosphono Y is a carbohydrate residue, are particularly effective in inhibiting the adhesion phenomenon.

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. (Wheeler, ef al., J. Clin. Invest. , £2:1211 , 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.

EXAMPLE 1

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 Berliner, ef al. (Arteriosclerosis, 6:254, 1986). Monocytes were isolated by a modification of the method of Recalde (Fogelman, ef 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 lymphocytes were prepared from human plasma employing previously described methods (A. Boyum, Scand. J. Clin., 21:77-89, 1968; Van Epps, ef al., J.Immunol., 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 NaCl containing 0.01% EDTA. MM-LDL was obtained by storage of LDL at 4^βC for 3-6 months, by mild iron oxidation (Kosugi, ef al., J. Cell. Physiol., 120:311, 1987), by copper oxidation, or by enzymatic treatment.

In the copper oxidation method, LDL is dialyzed to remove EDTA, then treated for 3 hours with 5-1 OμM cupric sulfate in PBS at 37° C. The reaction is stopped by addition of 0.2 mM BHT and 0.3mM EDTA. The LDL is then concentrated to the desired volume and dialyzed against PBS containing BHT and EDTA and stored at 4 ^βC.

The use of enzymes to produce MM-LDL from LDL can be achieved using soybean lipoxygenase (SLO) in the presence of phospholipase A2 (PU^)- Both of these enzymes have been shown to be important in the in vivo oxidation of LDL. In preparing MM-LDL using SLO and PLA-, it was assumed that only 70% of the enzyme would bind to the carrier beads. In performing these studies, alf reagents were prepared in LPS-free water. In preparing the bead (CNBR-activated Sepharose 4B) for coupling with SLO, 0.125g of beads were swollen using 5ml of 1mM HCl for 30 min. In the case of beads for coupling with

0.096g beads were swollen in 3.8ml of 1 mM HCl for 30 min. In both cases, the beads were washed 5x in 15ml polypropylene tubes. For enzyme coupling, the beads were washed 6x with coupling buffer (0.1 M NaHC0₃, 0.5M NaCl, pH 8.3) and the appropriate amount of SLO (sigma L- 8383, 5mg/1ml coupling buffer) or PU^ (sigma P-6139, 500 units in 500ml coupling buffer) was added. The respective enzyme bead mixture was then mixed for two hours at room temperature on an end-to-end mixer, then centrifuged to remove the supernatant. Any remaining binding sites on the beads were blocked by addition of 0.1 M Tris-HCl buffer (pH 8) followed by mixing over night at 4^βC. The beads were then washed 6x with PBS and brought to a final volume of 1.68ml for SLO and 1.40ml for PLA, with PBS. This procedure yields beads having a final concentration of SLO of about 100 units/ml and PLA_g of about 0.25 units/ml.

In preparing MM-LDL using SLO and PLA,, 1ml of LDL (0.5mg/ml), 5,000 units/ml of SLO beads, 20 units/ml of PLA_g beads and 20μl of 50mM calcium chloride were combined to a total volume of 1.0ml with PBS. Tubes containing the LDL/enzyme bead mixture were incubated for 24 hours at 37 ° C, at which time an additional 5,000 units of SLO beads were added to the tube, followed by an additional 24 hours of incubation. Following the final incubation, the tubes were centrifuged and the supernatant removed. In order to prevent further oxidation, 230μM BHT and 250μM EDTA were added to the supernatant containing the MM-LDL.

These minimally oxidized lipoproteins contain 2-6 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 of TBARS 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 5 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 RAEC. Although it was more difficult to obtain LDL active on HUVEC, about

10 1/8 of the preparations were active. The dose of a particular MM-LDL preparation necessary for maximal activity varied from 5-200 μg protein/ml. In addition, the optimal concentration of a particular preparation was always higher for HUVEC than for RAEC. Therefore, for experiments described herein, dose response curves were performed and the dose inducing maximal binding

15 was used in all experiments.

For studies on cellular adhesion, RAEC and HUVEC were rinsed with serum free media and 2 x 10⁵ THP-1, or monocytes in RPMl 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

20 number of attached cells was determined visually or in some cases, by measuring radioactivity of ³H thymidine or ⁵¹Cr labeled THP-1 adherent to the monolayer (Pawlowski, et al., Proc.Natl.Acad.Sci. USA, 82:8208-8212, 1985). In the case of U937 and T-cell adhesion to HUVEC, ceils suspended in PBS were injected into adhesion chambers and allowed to interact with the

25 endothelial monolayer for 10 min. The chambers were then inverted for 500

seconds to remove unattached cells (Smith, etal., J. Clin.lnvest, 22:2008-2017, 1989).

Earlier studies indicated that RAEC treated for 4 hours with 1-5 μg/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.

Four hour pretreatment of HUVEC with MM-LDL at 100 μg/ml induced a 4.7-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 significantly 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 lymphocytes to HUVEC was examined. Pretreatment of HUVEC with MM-LDL (100 μg/ml for 4 hours) induced binding of the U937 cells, a monocytic cell line, but did not induce the lymphocyte binding to the endothelial cells; however, lymphocyte binding was increased by 4-fold with LPS (10 ng/ml) pretreatment (FIGURE 2). It is important to note that the higher concentrations of MM-LDL were used for these studies as opposed to previous studies. Because HUVEC are less sensitive than RAEC to the effects of MM-LDL, concentrations up to 500 μg/ml may be necessary for effects.

- EXAMPLE 2

IMMUNOCHEMICAL 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 μg/ml), and IL-1 (10 U/ml).

HUVEC in 96-well dishes were pretreated with medium containing MM-LDL (100 μg/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 ¹²⁵l-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 lgG3, crossreacted with the ELAM-1 protein immunoprecipitated with H187 (Bevilacqua, ef al., Proc.Natl.Acad.Sci.USA, 84:9238-9242, 1987); P1 B5, an lgG1, crossreacted with VCAM-1 protein immunoprecipitated by antibody 4B9 (Carlos, ef al., Blood, 76:965-970, 1990); and P3G1 , an lgG1 , crossreacted with ICAM01 protein immunoprecipitated by RR1.1 obtained from Boehringer Mannheim. Unbound antibodies were removed by washing. The cells were dissolved in 2N 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 , however, 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 to ICAM (R 6.5), ICAM-1 remained unchanged. This lack of induction of ICAM-1 , ELAM-1 , and VCAM-1 was shown on several different MM-LDL preparations and on HAEC as well as HUVEC where binding was simultaneously measured. Lack of VCAM-1 induction was tested 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 18 antibody (TS1/18) 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) (obtained from Anthony J. Valente, The Cleveland Research Institute, Cleveland, OH) were prepared according to previous methods (Ishikawa, et al., J. Immunol.Methods, 28:117-123, 1980). Endothelial cells were treated with 10 μg/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 μg/ml of antibody (TS1/18, provided from C. Wayne Smith, Baylor College of Medicine, Houston TX) for 30 minutes, then monocytes were added to MM-LDL treated or untreated cells. In these experiments, pretreatment of HUVEC with Fab fragments of polyclonal antibodies to FN or MCP-1 failed to block the increased binding induced by MM-LDL (FIGURE 4). In addition, it was shown by ELISA that FN production was not induced by MM-LDL.

EXAMPLE 3

BIOCHEMICAL CHARACTERIZATION

Treating RAEC for 2 hours with cycloheximide (1 μg/ml) and tunicamycin (4 μg/ml) prior to the addition of the MM-LDL caused a 60% reduction in monocyte binding (FIGURE 5). Under the conditions employed, cycloheximide 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 or HAEC with MM-LDL for 4 hours, the cells were rinsed with ice cold PBS. RAEC were fixed with 1% paraformaldehyde in 1M sodium cacodate for 20 minutes at 4^βC. HAEC were tested unfixed. To measure the effect of sugars, fixed cells were washed two times with ice cold PBS and incubated with sugars; lactose-1 -phosphate (L1P), maltose-1- phosphate (M1P), N-acetyl-glycosamine (NAG), fructose-6-phosphate (F6P), mannose-6-phosphate (M6P), and glucose-6-phosphate (G6P) and glucose- 1 phosphate (G1 P) at lO^M each for 30 minutes at 4" C or 37 ^β C before adding THP-1 cells. The adhesion assay was carried out in the presence or absence of the sugars. Of the tested sugars, tactose-1 -phosphate, maltose-1- phosphate, and N-acetyl-glycosamine blocked binding by 90-100% (FIGURE 6). Other sugars, including mannose-6-phosphate, fructose-6-phosphate, glucose-1 -phosphate and glucose-6-phosphate, did not inhibit binding to MM- LDL treated cells.

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, ef al., Blood, 70:1842-1850, 1987). These 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 to protein inase C[PKC] in that H7 effectively inhibits PKC and HA1004 does not) on induction 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. Briefly, RAEC or HUVEC were treated with MM-LDL or Lipopolysaccharide (LPS, 1 μg/ml, from E. coli strain 0111 :B4, List Biological Laboratories) for 4 hours. To label the cells with ^S 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 5 then lysed in 1% Triton X-114 in 10 mM Tris-HCl, 150 mM NaCl, PH 7.4, 1mM PMSF, 20 μg/ml pepstatin and leupeptin. The lysate was centrifuged at 12,000 g for 5 minutes at 4^βC. The supernatant was layered on a sucrose cushion, warmed to 37 ^βC for 5 minutes, and centrifuged for 3 minutes at 300 g at room temperature. The detergent phase was subjected to trichloroacetic acid

10 precipitation and was analyzed by SDS polyacrylamide gel electrophoresis (Laemmli, U.K., Nature [Lond.], 227:680-, 1970). In some cases, the isolated ^S methionine labeled membrane proteins were incubated with glycopeptidase F (2 U/ml) in 0.2 M NaP0₄, PH 8.6, 1.25% NP-40, and 0.01 M β-mercaptoethanol for 18 hours. The gels were fixed, dried, and subjected to

15 autoradiography on X-ray film.

Equal amounts of TCA precipitable radioactivity from treated and untreated cells were subjected to electrophoresis under reducing conditions. Increased incorporation of ^S 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

20 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 same S methionine labeled membrane preparation after treatment with glycopeptidase. Glycopeptidase treatment reduced the molecular weight of two of the induced

25 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 Na¹²⁵l, 20 mM glucose, and 8-12 uU glucose oxidase. lodination 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 lysed 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.

EXAMPLE 4

TUMOR CELL ADHESION TO MM-LDL TREATED ENDOTHELIAL CELLS

Initial studies showed that tumor cells bind rapidly to MM-LDL stimulated endothelial cells and that incubation at room temperature for 10-15 minutes allows for a low level of adhesion to unstimulated endothelial cells with a measurable augmentation of adhesion to MM-LDL treated cells for some tumor lines.

Endothelial cell monolayers in 48 well tissue culture plates were incubated for 4 hours at 37 °C with MM-LDL or with control medium. The MM-LDL was rinsed off and 1.2 x 10⁵ tumor cells were added to each well containing the monolayers. The plates were incubated at room temperature for 10 minutes and the non-adherent tumor cells were then rinsed off. The number of adherent tumor cells remaining was counted microscopically using a microgrid. The number of cells adherent to MM-LDL treated monolayers was compared to the number adherent to control untreated endothelial monolayers. Table 1 reports the data from 2 different adhesion studies using 2 different MM-LDL preparations. Results using the human monocyte cell line THP-1, which is known to be responsive to MM-LDL induced endothelial adhesion, was also included.

TABLE 1 Adhesion of tumor cell lines to endothelial cell monolayers'

EXP 1 EXP 2

Data represent results from duplicate samples and are expressed as tumor cells/microscopic field.

SKBR-3 AND MCF-7 are both breast cancer cell lines which have been derived from pleural effusions and grow as adherent monolayers in tissue culture. However, in the study they showed marked differences in their adhesion properties both to unstimulated and to MM-LDL treated endothelial cells. SKBR-3 has a relatively high base line adhesion to unstimulated endothelial cells and displays a 4 to 10 fold increased adhesion to MM-LDL stimulated cells. The SKBR-3 adhesion to MM-LDL stimulated endothelium is comparable to levels seen with the monocyte line THP-1. MCF-7, on the other hand, has very low levels of adhesion to either the control or stimulated endothelium. The DLD, HT-29, and 716-1 tumor lines are all derived from primary colon tumors. DLD, and HT-29 grow in adherent monolayers in culture and both show increased adhesion to MM-LDL stimulated ehdothelial cells over control ehdothelium, although at lower levels than was seen with SKBR-3 or THP-1. While most established colon and breast tumor cell lines grow adherent to tissue culture plates in culture, the 716 Colon Tumor line is nonadherent, growing in suspension in culture. This line also fails to show significant adhesion either to MM-LDL stimulated or control endothelium which may indicate the loss of an important adhesion ligand in this cell line.

These preliminary findings indicate that MM-LDL can induce endothelial cell adhesion for some tumor cell lines. The reasons for the variation in the pattern of adhesion in the 5 cell lines which have been studied so far is not obvious, but may suggest differences between primary versus metastic lesions, differences in tumor types, alterations induced by tissue culture, or other variables leading to metastatic populations.

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

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. The process of claim 2, wherein the lipoprotein is oxidized using a copper salt.

8. The process of claim 7, wherein the copper salt is cupric sulfate.

9. The process of claim 2, wherein the lipoprotein is oxidized using enzyme.

10. The process of claim 9, wherein in the enzyme is a lipoxygenase.

11. The process of claim 10, wherein the lipoxygenase is soybean lipoxygenase.

12. The process of claim 9, wherein the enzyme is phospholipase.

13. The process of claim 12, wherein the phospholipase is phospholipase A*.

14. 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.

15. The process of claim 14, wherein the LDL is oxidized by storage under physiologic conditions at about 4^βC for a period of from about 2 months to about 8 months.

16. The process of claim 14, wherein the LDL is oxidized using a chemical.

17. The process of claim 16, wherein the chemical is an iron salt.

18. The process of claim 17, wherein the salt is ferrous sulfate.

19. The process of claim 16, wherein the oxidizing chemical contains from about 1 to about 10mM of TBARS/mg cholesterol.

20. The process of claim 14, wherein the lipoprotein is oxidized using a copper salt.

21. The process of claim 20, wherein the copper salt is cupric sulfate.

22. The process of claim 14, wherein the lipoprotein is oxidized using enzyme.

23. The process of claim 22, wherein in the enzyme is a lipoxygenase.

24. The process of claim 23, wherein the lipoxygenase is soybean lipoxyganase.

25. The process of claim 14, wherein the enzyme is phospholipase.

26. The process of claim 25, wherein the phospholipase is phospholipase Am,.

27. 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.

28. The method of claim 27, wherein the EMAM receptor is on an endothelial cell.

29. The method of claim 27, wherein the EMAM ligand is on a monocyte.

30. The method of claim 27, wherein the EMAM receptor is detectably labeled.

31. The method of claim 27, wherein the EMAM ligand is detectably labeled.

32. 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.

33. The method of claim 32, 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.

34. The method of claim 33, wherein the antibody is monoclonal.

35. The method of claim 32, wherein the member is EMAM ligand and the member binding ok is selected from EMAM receptor, an antibody specific for EMAM ligand, and a lectin.

36. The method of claim 35, wherein the antibody is monoclonal.

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

38. The antibody of claim 37, wherein the antibody is polyclonal.

39. The antibody of claim 37, wherein the antibody is monoclonal.

40. The antibody of claim 37, wherein the member is the EMAM receptor.

41. The antibody of claim 37, wherein the member is the EMAM ligand.

42. 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.

43. The method of claim 42, wherein the member is the EMAM receptor.

44. The method of claim 42, wherein the member is the EMAM ligand.

45. 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.

46. The method of claim 45, wherein the EMAM agent inhibits adhesion between the EMAM receptor and the EMAM ligand.

47. The method of claim 45, 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.

48. The method of claim 47, wherein the inhibitors has the structure:

wherein R is hydrogen or phosphono; X is hydroxyl, amino, or protected amino; and Y is hydrogen or a carbohydrate residue, with the proviso that when R is phosphono, Y is a carbohydrate residue.

49. The method of claim 48, wherein the carbohydrate residue is σ-D- glucopyranosyl or β-D-galactopyranosyl.

50. The method of claim 47, 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 45, wherein the EMAM agent is therapeutically labeled.

52. The method of claim 51 , 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.

SHEET

53. The method of claim 45, wherein the pathology is selected from the group consisting of atherosclerosis, inflammatory disease, autoimmune disease, and malignancy.

54. The method of claim 53, wherein the malignancy is a lipid related tumor.

55. The method of claim 54, wherein the tumor is colon carcinoma or breast cancer.

56. The method of claim 45, wherein the administration is parenteral.

57. The method of claim 56, wherein the parenteral administration is by subcutaneous, intramuscular, intraperitoneal, intracavity, transdermal, or intravenous injection.

58. The method of claim 45, wherein the EMAM agent is administered in a solid phase matrix.

59. The method of claim 58, wherein the matrix is biodegradable.

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

61. 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.

62. The method of claim 61, wherein the EMAM agent inhibits adhesion between the EMAM receptor and EMAM ligand.

63. The method of claim 62, 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.

64. The method of claim 63, wherein the inhibitors comprise the partial structure:

wherein R is hydrogen or phosphono; X is hydroxyl, amino, or protected amino; and Y is hydrogen or a carbohydrate residue, with the proviso that when R is phosphono, Y is a carbohydrate residue.

65. The method of claim 64, wherein the carbohydrate residue is σ-D- glucopyranosyl or β-D-galactopyranosyl.

66. The method of claim 63, wherein the inhibitors are selected from the group consisting of iactose-1 -phosphate, maltose-1 -phosphate, and N- acetyl-glucosamine.

67. The method of claim 61, wherein the detecting is in vivo.

68. The method of claim 67, wherein the detectable label is a radioisotope or a paramagnetic label.

69. The method of claim 61 , wherein the detecting is in vitro.

70. The method of claim 69, 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.