JPH05507519A - Cell-cell adhesion mediator - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Cell-cell adhesion mediator field of invention The present invention reduces or suppresses inflammation, and the process of inflammatory diseases and intercellular connections. The present invention relates to compositions and methods for treating other pathological conditions mediated by cancer.

Background of the invention Vascular endothelial cells and platelets participate in certain biological responses in the bloodstream. plays an important role by selectively binding to cells, e.g. phagocytic leukocytes perform. For example, endothelial cells, monocytes and granulocytes attack blood vessel walls in response to inflammation. It binds preferentially to the tissues before moving through and into the surrounding tissues. a Some types of inflammation-triggering compounds act directly on the endothelium of blood vessels, causing whitening of the blood vessel walls. It is known to promote blood cell adhesion, which then allows cells to pass through walls and be damaged. or move into an infected area. Adhesion of cells to the endothelium of blood vessels also contributes to tumor metastasis. It is thought that this is related to Circulating cancer cells apparently interact with the body's normal inflammatory mechanisms. and bind to areas of the blood vessel wall where the endothelium is activated.

Platelets are also involved in similar responses. Platelets are activated during the onset of hemostasis. It is known that functional changes (e.g., rapid granule exocytosis or degranulation) and At this point, the alpha granule membrane of the platelet becomes fused with the external plasma membrane. the result, New cell surface proteins are now expressed, and these New functions of platelets, such as binding to both other activated platelets and other cells. give the ability to match. Activated platelets are recruited into the growing thrombus or or rapidly removed from the blood circulation. Activated platelets are linked to monocytes and It is known to bind to phagocytic leukocytes, including neutrophils. This process Examples of pathological and other biological processes mediated by atherosclerotic Including arteriosclerosis, blood clotting and inflammation.

In recent studies, specialized cell surface receptors on endothelial cells and platelets -, endothelial leukocyte adhesion molecule (ELAM-1) and granulosa protein, respectively. 140 (GMP-140), which is designated as It was revealed that this is related to the recognition of cells. These receptors Chin-like domain, region with homology to epidermal growth factor, and complement tII control It is a region with homology to proteins (see, Bevi 1acqua et al., 5 science243:1160 (1989), which is incorporated herein by reference. ). For example, ELAM-1 is the first step in many inflammatory responses. It is known to mediate endothelial leukocyte adhesion. Specifically, ELAM-1 is human Neutrophils, monocytes, eosinophils, and certain T lymphocytes (N, Graber et al., J. [m munol, 145:819 (+990, NK cells and promyeloid cells) It binds to the cell line HL-60.

The term "selectin" refers to receptors, including ELAM-1 and GMP-140. A general class of tars has been suggested.

Because their receptors have lectin-like domains and their attachment This is because functions have a selective nature. These cell surface receptors are expressed on cells. GMP-140 (also known as PADGEM) is present on the surface of platelets and endothelial cells, where it is present on the surface of platelets-leukocytes and Mediates endothelium-leukocyte interactions. Other members of the selectin class are ME L-14 antigen and its human analogue LAM-1, which are found in lymphoid cells. surface receptors and act as lymph node homing receptors. Ru. However, the exact nature of the ligands recognized by selectin receptors The quality remains largely unknown.

In order to block the action of selectins and thus inhibit cell adhesion, Various other methods have been developed in the past. For example, for ELAM-1 The use of monoclonal antibodies directed to the treatment of pathological responses, e.g. It has been proposed as a method to inhibit endothelial-leukocyte adhesion. endothelial i Interleukin-8 has also been shown to be an inhibitor of leukocyte-endothelial cell interactions. is known.

Elucidation of ligand-receptor interactions may be useful in therapeutic regimens. The goal is to develop highly specific and efficient inhibitors of selectin-mediated cell adhesion. You can do something like that. Also, 1st type or 2nd floor! Pharmaceutical compounds using more than one ligand targeting agents, e.g. anti-inflammatory agents or antioxidants, to the site of injury. Can be done. However, to date, the ligands on Ifi or 2FipI and These efforts are hampered by an insufficient understanding of the interactions of receptor molecules and receptor molecules. is interfering with The present invention satisfies these and other related needs.

Summary of the invention According to the invention, selectively binding to a selectin receptor and at least one Novel compositions having oligosaccharide components are provided.

The selectin-binding component has the formula: %formula%() R3 is an oligosaccharide or R, -R, -C(CO2H)-, and R3 and R4 are are the same or different, and -H1-CI -C8 alkyl, -hydro RoxylCl-C8 alkyl, -arylC1-C8 alkyl, or -alco is xyCl-C8 alkyl, R2 is βI, 3Gal, αl, 2Man, or αl, 6GalNac, and a is 0 or 1] has.

In certain preferred embodiments, R1 is sialic acid, typically NeuAC or Neu It is Gc. When R6 is an oligosaccharide, it is preferably NeuAca2.3 Galβ1.4GIcNAcβl,3, or NeuGca2.3GalβI, 4G1cNAcβ1,3. Selectin-binding components typically include selectin-binding components. Contains the smallest tetrasaccharide (referred to as SLX) recognized by the receptor. It is an oligosaccharide consisting of

The compound typically does not contain an unfucosylated SLX core structure. It is prepared from a polysaccharide with repeating units. When fucosylated, polyvalent SLX A polysaccharide having the following properties is obtained. Type Ia, IV and IN group B strains preferred for this purpose. It is a polysaccharide from Leptococcus. polyvalent Selectin-binding compounds also include selectin-binding moieties as part of various linkers. Obtained by chaining.

The claimed compositions provide a method for treating cells mediated by selectin cell surface receptors. This inhibits adhesion between cells, thereby inhibiting, for example, inflammation and other adhesion-related Pathological responses can be inhibited.

In a related embodiment, the composition that binds a selectin is a glycoprotein, a glycoprotein. It can be a pido or an oligosaccharide.

The invention particularly provides the above compounds in pharmaceutical compositions. Pharmaceutical compositions include e.g. For example, ingredients and drugs that can selectively bind to selectin receptors. The liposome may comprise a carrier that is acceptable as a pharmaceutically acceptable carrier. this Liposomes with components can also be targeted for common chemotherapeutic agents. Acting as a vehicle, the chemotherapeutic agent is encapsulated within liposomes and selectively The receptor is supplied to the collared cells expressing the receptor. Typically, chemotherapy The agent is an anti-inflammatory factor or an antioxidant. Chemical factors encapsulated within liposomes The use of the ingredients described herein to target It is a convenient and effective way to lower legal levels and minimize side effects.

The pharmaceutical composition of the present invention also contains oligosaccharides recognized by selectin receptors. can comprise an immunoglobulin capable of selectively binding to a ligand; Wear. Immunoglobulins suitable for this purpose include C3LEX~1. FH6, SNH, , SNH, and VIM-2.

In another aspect, the invention provides for administering to a patient a therapeutically effective dose of a selectin receptor. by administering a compound comprising a moiety capable of selectively binding to to inhibit adhesion between cells that cause disease processes, e.g. inflammation, in patients. including methods of harm. Cell surface receptors such as ELAM-1 or GMP -140 can be expressed on vascular endothelial cells or platelets. Inflammation For example, septic shock, sepsis I! I continuous wound, acute breathing It is.

Brief description of the drawing Figure 1 shows expression of lr-cyfulized Le” (CHO-kl and LEC12). selectin (L) that binds to IL-1β-activated endothelial cells compared to EC11).

Figure 2. (Fig. 2A) and 4°C (Fig. 2B) on uNrHL-60 cells. Ability of monoclonal antibodies specific for SLX to block SLX-mediated binding shows.

Figure 3 shows SLX and non-SLX specific binding to activated endothelial cells. LECII (Fig. 3A) and LEC12 (Fig. 3B) with specific monoclonal antibodies. ) shows the effect of cell incubation.

Figure 4 shows HL-60, LECll and L before binding to activated endothelial cells. The results obtained by treating ECI 2 cells with sialidase are shown.

FIG. 5 shows SLX, which inhibits the binding of HL-60 cells to activated endothelial cells; The ability of liposomes containing glycolipids with a similar carbohydrate structure to Compare power.

Figure 6 shows that monoclonal antibodies specific for SLX and Le'' determinants and GMP-140-mediated inhibition of platelet adhesion.

Figure 7 shows that SLX, L inhibits binding of HL-60 cells to activated platelets. Ability of liposomes containing glycolipids with "e" or similar carbohydrate structures Compare.

Figure 8 shows SLX, Le” or compared the ability of liposomes containing glycolipids with similar carbohydrate structures. Ru.

Figure 9 shows the results of glycolipids with terminal sialic acid NeuAc or NeuGc. Figure 2 shows inhibition of GMP-140-mediated adhesion.

Figure 10 shows monoclonal antibodies against ELAM-1 administered prophylactically. Shows to prevent ribopolysaccharide induction in rats.

FIG. 11 shows a therapeutically administered monoclonal antibody against ELA, M-1. shows that the drug prevents ribopolysaccharide induction in rats.

Description of preferred embodiments and compositions that inhibit inflammation and other disease responses mediated by cell adhesion. A method is provided. The present invention also provides for Compounds that develop the ability to block or inhibit cell adhesion (e.g., glycoconjugate) conjugated and monoclonal antibodies). How to prepare such compounds is also provided. Additionally, diagnostic and therapeutic uses of the compounds are provided.

The basis of the invention is that carbohydrate moieties recognized by selectin cell surface receptors This is the discovery. As mentioned above, selectins also promote the cell adhesion molecule rLEC. - Unique sugar proteins expressed on the surface of various cells, known as the CAMJ family. It is protein. For example, ELAM-1 is inducibly expressed on vascular endothelial cells. (Bevilacquaet al., supra and He5sion et al., Proc. , Natl. Acad. Sci., 87:1673-1677 (1990) , both of which are included here by reference). This receptor is an inflammatory receptor. Tocains, such as interleukin β (IL-rβ) and tumor necrosis factor α (TNFα), as well as bacterial endotoxin (ribopolysaccharide)-induced It has been proven that (see Bevi Lacqua et al., Proc. Natl. Acad, Sci., 84:9238-9242 (1987), here (added by citation). These compounds act directly on endothelial cells in vitro. enhances the adhesion of polymorphonuclear leukocytes (neutrophils) and monocytes (Bev i Iac qua et al., Proc. Natl., Acad. Sci, supra).

As mentioned above, GMP-140 is a membrane protein of platelets and endothelial secretory granules. (Geng et al., Nature, 343.757-760 (1990 ), which is added here by citation). releasing GMP-140 on their surfaces. Activated platelets bind to monocytes and neutrophils.Jungi et al., Blo. od 67:629-636 (1986)), and also monocytoid cell lines, For example, it is known to bind to HL60 and U937 (Jungi et al. , supra; 5ilberstein et al., J. Cl1n, (nvest, 79: 867-874 (+987)), all of which are incorporated herein by reference.

GMP-140 is an alpha granulosa protein with a molecular weight of 140.000 and is Expressed on the surface of activated platelets during platelet stimulation and granule secretion ( Hsu Lin et al., J. Biol. Chem., 259:9121-9126 (1984): It was done. Furie et al., U.S. Pat. No. 4,783,330 , describes monoclonal antibodies reactive with GMP-140. Reference text above All contributions are included here by citation.

The third selectin receptor is the lymphocyte homing receptor, MEL-14. antigen or LAM-] (Gallatin et al., Nature 304:30 -34 (1983); Siegellman et al., 5science 243 :11651172 (1989);-1 plays an early role in the binding of neutrophils to the endothelium. I believe it works.

The structure and function of the selectin luceptors are elucidated by cloning and expression of long cDNAs (see, e.g., Be vHaequa et al., 5science, 5upra, (E LAM-1) , Geng et al., supra (GMP I 40), and La5ky et al., supra (M EL-14t'ic original)). The extracellular component of selectin is a previously described protein. It can be divided into three segments based on homology to the protein. N-terminal region (approximately 120 amino acids) contains the low affinity IgE receptor CD23. ickamer,, 1Bio1. Chem, 263:9557-9560 (1988) (which is added here by citation) Related to the mammalian cutin protein family. A segment of the polypeptide follows. This is related to proteins containing epidermal growth factor (EGF) motifs. It has an array. Finally, after the EFG domain, one or There are two or more tandem repeat motifs that are found in the family of complement regulatory proteins. Concerning what exists.

Selectin receptors contain lectin-like domains, so these molecules Specificity appears to be based on protein-carbohydrate interactions. provided here Evidence suggests that Lewis X antigen (here designated SLX) The sialylated and fucosylated N-acetyllactosamine units of This is the part recognized by the lectin region of Sebuta. In particular, the evidence is ELAM The recognition of this part by both GMP-1 and GMP-140 is shown.

The compositions of the present invention contain this sialylated, fucosylated N-acetate in various configurations. It comprises lulactosamine units.

Selective binding, as used herein, refers to spatial or polar binding of determinant sites on a second molecule. Sexual 8! one molecule (typically called a receptor) based on the structure of another molecule (typically called a receptor) refers to the specific recognition of a child (typically called a ligand). Choice between two molecules binding occurs when the affinity is sufficiently strong. Binding affinity is typically defined as association and the affinity constant (K, ) for the equilibrium concentration of the dissociating configuration. Ru. That is, K, = (RL)/(R) (L), where (R), ( L) and (RL) are receptor (R), ligand (L) and is the concentration at equilibrium of the receptor-ligand complex (R-L).

The specific binding interaction of receptor and ligand molecules is typically reversible. non-cooperative associations, e.g. electrostatic attraction, van der Waalska and hydrogen is a combination (see generally, 5tryer.

BiochemiStry (W, H, Freeman and Company , New York, 3rd edition, +988), incorporated herein by citation in its entirety. ). Examples of selective binding include antibody-antigen recognition, enzyme-substrate recognition, and the like.

The nomenclature used to describe the oligosaccharide components of the present invention follows conventional nomenclature. Individual Use standard abbreviations for each monosaccharide. For example, 2-N-acetylglucosa Min is converted to GlcNAc, fucose is converted to Fuc, and galactose is converted to Gal. , and glucose is represented by Glc. What is present in the oligo spear beads of the present invention? The two sialic acids that can be and 5-N-glycolylneuramic acid (NeuGc).

Unless otherwise specified, all sugars, with the exception of fucose (L-isomer), are cyclic pyranoses. It is the D-isomer in the S configuration. The two cyclic anomers are separated by α and β. Represent.

Monosaccharides are generally linked by glycosidic bonds to form oligosaccharides and polysaccharides.

The orientation of the bonds with respect to the plane of the ring is indicated by α and β. bond between two monosaccharides The specific carbon atoms that form are also listed.

Thus, the β-glycosidic linkage between C-1 of galactose and C-4 of glucose The case is represented by GalβI, 4G1c. D-sugar (e.g. D-G lc NAc, D-Ga 1 and D-NcuAc), the display α is C-1 (N The hydroxyl attached to C-2) in euAc is below the plane of the ring. and β means on the ring. Odor in case of L-fucose , the designation α means that the hydroxyl is present on the ring, and β means that it is means below.

Carbohydrates through which SLX mediates leukocyte-endothelial cell and leukocyte-platelet cell adhesion SLX or its pseudo-SLX (mimet Compounds comprising ic s) can be produced from tfJ or newly produced. It can be synthesized. As described below, the present invention provides selectin-binding properties of the present invention. A variety of compounds are provided comprising the following ingredients: For example, a biological molecule having the component It can be used as a compound. The biological molecules defined here are: Including, but not limited to: molecules of biological significance, e.g. , amino acids (and their mimetics), oligopeptides, proteins (e.g. glycoproteins and proteinaceous hormones), fatty acids, lipids (e.g. pids, phospholipids, sphingolipids and gangliosides), steroid hormones oligosaccharides, polysaccharides, and nucleic acids (e.g., deoxyribonucleic acids and ribonucleic acids) ). These compounds can be purified and/or processed according to standard techniques known to those skilled in the art. or can be synthesized. In addition, a wide variety of compounds containing this component, As described below, it can be newly synthesized.

Once obtained, such compounds can e.g. for a variety of purposes, including competitive inhibition of the binding of SLX-bearing cells to expressing cells. can be used. Binding a compound of the invention to a cell surface selectin The interaction of selectins with native SLX on migrating cells is prevented by Normal and pathological binding of leukocytes and other cells to endothelium or platelets is disrupted . Thus, compounds containing one or more SLX units or pseudo-SLX are For example, inflammation, allelosclerosis, coagulation and other endothelial or platelet-mediated It can act as an effective inhibitor of pathological events.

Sialic acid residues in SLX are It can be of different forms. Typically, sialic acid is 5-N-acetylneu Lamic acid (NeuAc) or 5-N-glycolylneuramic acid (NeuGc) It is. However, other sialic acids can be used in their place . An overview of the different forms of sialic acids suitable in the present invention generally include R,5cha ur.

Methods in Enzymology, 50: 64-89 (1 987), and 5chaur, Advancesin Carbohydra te Chemistry and Biochemistry 40: 1 See 31-234. Both of them are included here by citation. lower fruit As shown in Example ■, the affinity of selectin receptors is are identical if the oligosaccharide ends in NeuGc. In this way, the use The term "SLX" as used herein refers to the smallest tetrasaccharide unit (sialic acid α2.3Galβ1. 4 rFucaL3)Glc(β1,3)), where sialic acid is NeuAc, NeuGc or other equivalent form of sialic acid. be.

In addition to these structures, the sialic acid residue is shown as NeuAc. It is understood to include forms of NeuGc, in particular NeuGc.

The evidence provided below or as shown in the formula: %formula% Pentasatucaride, which contains It is the smallest structure to grow. Thus, in certain preferred embodiments, the oligosaccharide is It contains this pentasatucharide structure.

Other changes in the basic SLX unit are also recognized by selectin receptors. be done. For example, the evidence provided in Example ■ below shows that the structure NeuGca2 ．． 3Galβ1, 4G1cNAcβ1.

3Ga IβI, 4 (Fucαl, 3) G1cNAcβ1, 3Galβl, 4 An oligonucleotide called SY (also known as VIM-2 antigen) that grows GIcβ. The gosaccharide component has been shown to bind to selectin receptors as well as SLX.

The SY2 moiety comprises two sialylated N-acetyllactosamine units, which One is SLX. Thus, oligos recognized by selectin receptors Sugars consist of a large number of sialylated N-acetyllactosamine units, the least of which The other is fucosylated (see Teimeyer et al.

Proc, Natl, Acad, Sci, (USA) 88: 1138-1 142 (1991), incorporated herein by reference).

The oligosaccharide component of the invention preferably terminates in a sialic acid residue. some kind of In embodiments, the sialic acid residue further comprises another saccharide residue, e.g. a second sialic acid residue. can be linked to a C2,8 chain to a C2,8 chain.

Alternatively, the terminal sialic acid residue can be substituted with various groups. thus , certain selectin-binding components of the invention have the formula R, -NeuAcα2,3C ;alβ1.4GlcNAcβ1- (where R1 is R3R4C(CO2H)-, and R3 and R2 are the same or However, H1 lower alkyl (CI-C8), hydroxyl lower alkyl (C1-C8), arylalkyl or alkoxyalkyl) Ru. Furthermore, R2 and R8 are connected to form a 4- to 8-membered carbocycle or heterocycle. be able to.

Compounds containing SLX and related structures appear on the cell surface of many cell bodies. It can be obtained from proteins or glycolipids.

For example, the SLX antigen is expressed on the cell surface of LEC11 cells and CHO cells with glycosylation mutations. It is present in the N-linked carbohydrate base of facet proteins. LECll is SLX This unique protein contains terminal structures that support both sialic acid and fucose within its sequence. Expresses specific glycoproteins where R is: Add this by citation. ) Using the procedure described below, the LECII mutants were It has been shown to bind to activated human vascular endothelial cells. Wild type CHO cells and other related glycosylation patterns that do not have a specific glycosylation pattern (SLX). The mutant CHO cell line showed the same level of binding.

Other sources used to obtain SLX units are glycolipids or glycoproteins. Includes any cell that expresses a component of a carbohydrate matrix. Thus, polymorphonuclear neutrophils This unit has been purified using , lymphocytes, tumor cells or HL-60.

Other cells that bind to activated vascular endothelial cells can be used to separate the ligand. (see Symington et al., J. Immunol, 134: 2498-2506 (1985).

Llizoguchi et al., J. Biol. Chem., 259:11949- 11957 (1984), Mizoguchi et al., J. Biol, Chem. , 259:11943-11948 (1984), Pa1etta et al., C. ancerRes, 48:28-287 (1988). Add this by citation.

Compounds containing SLX or its pseudo-SLX are surface proteins, glycopeptides, , commonly used in this field for separating oligosaccharides and glycolipids from cells. It can be prepared from natural sources using known methods (see, e.g. Gerard, Purification of g! 1coproteins ) and Thomas et al., rPurification of membrane neproteinsl, both Guide to Protein Pur ification, Vol, 182゜Methods in Enzym (edited by Deutscher, 1990), which is quoted here. ). For example, using LECI+ cells, eg, 5tanleY et al. Glycoproteins and glycolipids containing SLX units were prepared using the methods described above. can be obtained. Briefly, in one method, LECI! cells Infection with vesicular stomatitis virus. Then, the structural The carbohydrate modification is due to the N-linked biantennary (bian) of the viral G-glycoprotein. expressed on carbohydrates (tenary). This virus was isolated by equilibrium gradient centrifugation. purified and glycopeptides were purified as described by Tanley et al. Purify by teinase digestion.

Using several approaches, HL-60, HT-29°colo205, Neutrophils and other cells containing ligands recognized by selectin receptors Isolate the selectin-binding moiety from the cell system. Ligands generally target these cell types. Since it is expressed on the cell surface, one approach is to isolate the ligand-enriched plasma membrane fraction. comprising separating the Once the plasma membrane is separated, the ligand can be separated Subsequently, monoclonal antibodies, especially those reactive with the SLX oligosaccharide structure, , for example using monoclonal antibodies FH6,5NH3 and C3LEX-1. It can be identified by

Glycopeptides were used to characterize selectin receptor ligands on glycoproteins. The release of the oligosaccharide from the tide is the first step in the structural analysis of the oligosaccharide chain. This is by chemical cleavage of protein-carbohydrate chains or by endoglycosidases. This is achieved by liberating oligosaccharides. is different in most cases Precise conditions can be established for individual glycoproteins using procedures . Asparagine-linked oligosaccharides are involved in hydrazitolysis, endoglycosidase, and released by alkaline hydrolysis and trifluoroacetolysis. 0 consecutive Chain carbohydrate positions are released by alkaline β-elimination. Gel filtration of oligosaccharides separated from glycopeptides. Next, the resulting oligosaccharides were subjected to gel filtration and HPLC. 1 Combination of thin layer chromatography and ion exchange chromatography Use to separate them from each other. The separated oligosaccharides are then thoroughly analyzed. refined Analysis of the complete structure of the oligosaccharide units consists of monosaccharide units, their ring shape, configuration (D or or L), anomeric linkage (α or β), position of linkage between sugars and their sequences. Requires determination of location.

Additionally, the positions of any substituents are established. Methylation is used to link groups between monosaccharides. Determine the position of the lycoside linkage. The anomer configuration of sugar residue is 500-λIH can be addressed using NMR spectroscopy of z'H. complete structure Conditions and methods used to perform carbohydrate analysis are described by Beeley, Labor atory Techniques in Biochemistryand Molecular Biology, Burdon and Knippenb Generally described in erg, Matoi, Elsevier, Amsterdam (1985). and is included here by reference.

The state of the art for fully characterizing the sugars of oligosaccharides is based on several analytical techniques. , for example, FAB-MS (Fast Atom Collision Mass Spectrum), HPAE (High pH anion exchange chromatography) and 'I (-NMR) Ru. These technologies are complementary. Structure of oligosaccharides using these techniques A recent example of a method to fully characterize the , J. Biol, Chem.

264:14100 (+989), and 5tanley et al., supra. I can only give it out. Another method is positive ion fast atom collision mass spectrometry (FAB- MS) and gas chromatography-quadruple mass spectrometry (GC/E I-M S) (see EPO application no. 89305153.2) No., incorporated herein by reference).

One approach to characterizing selectin ligands on glycolipids is to was disrupted with an organic solvent, the glycolipid was separated, and the monochrome against SLX null antibodies, such as FH6,5NH3゜5NH4, C5LEX-1 or VIM -Identifying the glycolipid reactive with 2 and then determining the structure of the oligosaccharide chain. Consists of.

To obtain glycolipids and gangliosides containing SLX, glycolipids Standard methods of preparation can be used (see, e.g., Ledeen et al. , J. Neurochem, 21:829 (1973), this here (add by citation). For example, glycolipid HL-60, HT-29, PM N., human leukocytes, and Ike cell lines expressing selectin ligands. extraction by methods known to those skilled in the art (see, e.g. Symington et al., J. Immur+o1. , +34:2498 (+985) and kla cher and Beckstead, [, euemia Res, 14: 119-130 (1990)).

Cells! ! The cells are grown in suspension and harvested by centrifugation. Glycolipid, K annagi et al., J. Biot, Chem., 257:14865 (+9 82) (incorporated herein by reference). Chloroform/methanol 2,1 and isopropyl alcohol/hexane from the precipitate Extract with sun/water 55:25:20. The resulting extract was dissolved in chloroform/ Partition with methanol/water (3:2+1). The upper phase of the resulting extract is It contains lioside and the lower phase contains glycolipid.

Gangliosides (glycosphingoli containing at least one sialic acid component) Ledeen and Yu, AIethOdSEnz ymol, 83:l39(+982) (by reference here). (incorporated into the specification) and DEAE-Sephadex ( S e p h a d e x) Neutral and acidic by chromatography Divide into fractions. The resulting gangliosides are pooled, lyophilized, and chloro Dissolve in form/methanol (2:1).

The lower phase of the Folch partition contains glycolipids. separate these , and preparative thin layer chromatography using chloroporum/methanol/water (60 :35:8) as the solvent system. Separate as shown.

Identifying gangliosides and glycolipids containing selectin ligands For immunochemical glycolipid analysis iAIgnai et al., Analyt, Bi ochem, 109:399 (1980) (incorporated herein by reference). Implement according to the procedures described in Briefly, the aforementioned ganglioside pool is chromatographed by thin layer chromatography. Then a thin layer of Incubate the rate with 1211-labeled FH6, or related constructs. . After incubation with the labeled antibody, the plate is placed in a radiograph detection filter. Expose the film and develop it. Black spots on X-ray film are monochrome corresponds to the gangliosides that bind to null antibodies, and those gangliosides , scrape the corresponding area of the silica plate, and chloropholate the gangliosides. Collected by elution with lum/methanol/water. Gangliosides again dried and resuspended in chloroporum and developed in a similar thin layer system, and blobbing with a radiolabeled antibody. Glycolipid-derived Structural analysis of oligosaccharides is performed essentially as described for gangliosides. .

Oligosaccharides comprising SLX units can be prepared by methods known in the art. (see, eg, Gerard, supra, p. p, 537-539), typically N-glycosidase (N-glycanase ) is used to cleave the N-one-linked oligosaccharide, while the 〇-one-linked group is endo-N-acetylated. Cleave with lugalactosaminidase.

Synthetic compounds containing SLX or its pseudo-SLX bound to various components can be can be prepared depending on the specific application. For example, SLX is a ceramide part gangliolysis by linking C-1 of the GIcNAc unit at the reducing end. It can be converted into de. SLX structures and related structures are also available in a wide variety of other molecules, such as various substituted amino acids, heterocyclic compounds, branched or linear ether bond with a carbon chain, and an aryl or alkylaryl moiety. Can be chained to ether bonds with Selectin-binding components also have various Polysaccharide layer, amino acids, amino acids, oligopeptides or proteins, The binding can be carried out by methods well known in the art.

The term "alkyl J" as used herein refers to a branched or straight chain saturated or unbranched Saturated hydrocarbon chains, e.g. lower alkyl containing 1 to 7 g carbon atoms, e.g. , methyl, ethyl, n-propyl, butyl, n-hexyl, etc., cycloalkyl (3-7 carbons), cycloalkylmethyl (4-8 carbons) and arylalkyl means.

The term "aryl" refers to derivatives derived from aromatic hydrocarbons by removing one atom. means a derived radical, for example phenyl from benzene. Aromatic hydrocarbons are 1 It can have more unsaturated carbocycles, for example naphthyl. for#! 'a "Lukoxyl" refers to an alkyl group attached by an oxygen to the remainder of the molecule, e.g. means syl group. "Heterocycle" means a ring having 3 or more atoms. in which at least one of the atoms is other than carbon (e.g. N, O, S, Se, P or As). Examples of such compounds are furans, pyri Includes midines, purines, pyrazines, etc. for! ! "Oligo" means 2~ Molecules of polymers consisting of IO residues, e.g. amino acids (oligopeptides), monomers Refers to sugars (oligosaccharides) and nucleic acids (oligonucleotides). The term "poly" means a molecule of a polymer consisting of more than 10 residues.

Containing SLX or other structures for the synthesis of multivalent forms of selectin binding components from 1 to about 4 or more selectin binding moieties by combining units of monomers that can form molecules with

One example of such a polyvalent form is that the unit has the following components: n and m are the same. or different, and is an integer from 2 to 12, Y is O or S, and W is OlS or NH) It is linked by Alternatively, the moiety is a 5- to 14-membered ring with two substituents. and each substituent has the formula %formula%) and the substituents are in a cis or trans relationship.

SLX and related structures also include various heterocyclic compounds (e.g., containing nitrogen atoms). can be combined with In this case the ingredients are preferably Bonded to the nitrogen atoms of the ring, each nitrogen linked to one component. suitable for this purpose Examples of peterocycle compounds include piperazine and homopiperazine.

Alternatively, multivalent forms of SLX or l1fSLX can combine multiple bonds of the desired moiety. can be made by bonding to a carrier component formed by a front plate with a . Examples include SLX, lysine, lysine-containing peptides, proteins, and glycoproteins. Binding to the amino group of the asparagine side chain of proteins or such compounds can be mentioned.

Another method for preparing multivalent selectin-binding compounds of SLX is to attach the desired monosaccharide residues to It is added to polysaccharides. For example, a repeating unit with a linear core structure of SLX ( In other words, polysaccharides containing polysaccharides (which do not have fucose residues) are processed by enzymatic fucodylation. It can be converted into a polysaccharide containing polyvalent SLX. Group B streptococci Preference is given to using natural polysaccharides of type Ta, II or type II obtained from Many of these Type II, ^TCC No. 31577) can be isolated by standard procedures. Ru. For example, Jennigs et al., Biochemistry, 221258 -1263 (1983) and PCT Application Publication No. 8706267. and. Both of these are included here by citation.

These polysaccharides consist of repeating units of the formula:

The arrows in the structure above indicate the backbone of each polysaccharide molecule. As if buried, type 1a is Contains repeating units with a linear core structure of SLX. The two polysaccharides in the pond are the main chain comprises the core structure of SLX. α1,3-F according to the standard technique described below. Enzymatic fucosylation of these polysaccharides using cosyltransferases yielding the SLX compound. After fucosylation, the repeating unit is an Ia structure with the following formula: Generate the next intermediate. The transglycosylation reaction is optimally performed using an alkaline phosphatide. addition of atase (e.g., from calf intestine, CIAP) to inhibit the reaction. There are nucleoside phosphate by-products that can be consumed.

General conditions are known for enzymatic synthesis for the preparation of carbohydrate groups similar to SLX. (see T'oone et al., Tetrahedron 45:5365-5 422 (1989); Wong et al. J. Am. Chem. Soc. 47:5 41f3-5418 (1982): Added by Unverzagt).

Each of the major enzymatic reactions is shown (added by Beyer et al. ), galactosyltransferase and! for preparative reactions. or 2 The above sialyltransferases are purified from natural sources (Beyer et al., supra). and Weinstein et al., J. Biol. Chem, 257-21383. 5-13844 (1982), incorporated herein by reference). Fukoshi Transferases can also be determined by CrawIey. gala The DNA of ctosyltransferase and sialyltransferase was cloned. (Paulson and Colley, J., Biol, C. hem, 264:17615-17618 (1989), which is cited here. production of soluble recombinant enzymes for large-scale preparative synthesis. (Colley et al. 1. L Biol, Chem, 264:176 19-1.7622 (1989)).

In order to obtain sufficient amounts of fucosyltransferase for large-scale reactions, Some manufacturers have cloned this enzyme and are using it as a recombinant soluble enzyme. It can be expressed as A preferred method is Chirgwin et al., Bio Chemistry 18:5214-5299 (1979) RNA was extracted from wild-type CH('J and LECII cells) and and polyA+RNA by oligo(dT)-cellulose chromatography. Can be separated. Next, cDNA from LEC-11 cells was transferred to Sambr ook et al., Molecular Cloning: A Laboratory M annual, 2nd edition (1989), Co1d Spring Harbor Press, New York (which is included here by citation). It can be prepared as follows. eDNA was developed by Davis (Handboo k of ε experimental Immunology, Vol, 2. pp , l-13(+986) from wild-type CHO cells by the method of +RNA can be used to extract the CHO cells with the desired fucosyltra does not express spherase, but otherwise the mRNA FII of LECII cells Most of them exist. Next, extract the cDNA library into the DM8 expression vector. It can be constructed using the released cDNA (Seed, Nature 329:840-842 (1987 houses. Fucosyltransferase expressing The clones described in Larsen et al., Proc, Natl, Acad, Sci, 8 By expression cloning described in 63227-8231 (+989), using transfection of CO3-1 cells and FH6 antibody or SLX Screening cells expressing the SLX antigen with other antibodies specific for the antigen It can be separated by Then, the fucosyltransferase Using full-length clones, as taught by Coley et al., supra. and can produce soluble recombinant enzymes.

Another source of SLX is α1-acid glycoprotein, which is a plasma protein. and its carbohydrate moieties can be fucosylated to produce SLX (see, Alpha, -Acid glycoprotein: Genetics, B iochemistry, Physicological Function , and Pharmacology.

Bauman et al. (Wiley 1989), and Walz et al. , 5science 250:1132-1135 (1990), those (both are included here by citation).

Enzymatic synthesis or combined chemical and enzymatic synthesis of SLX compounds is preferred. However, chemical synthesis can also be performed as shown in reaction schemes ■ and ■a below. It is possible.

Scheme If Scheme II a Enzymatic synthesis or combined chemical and enzymatic synthesis of SLX compounds is preferred. However, chemical synthesis is also possible as shown in Reaction Summary ■ and IIa below. It is Noh.

The component pieces of the SLX structure are synthesized. For example, containing sialic acids, including SLX. The glycoside having (Added by reference). N1coLaou et al. (J, Am, Ch Em, Soc, 112:3693 (1990)) is a glycosphingolipid. presented a complete synthesis of the Lex family associated with human tumors. As shown in scheme■ , trisaccharide Galβ1.4 (Fucα1.3)GlcNAc protected therein The synthesis of (A) is described. This intermediate and a suitable glycosyl acceptor ( For example, reaction with an alcohol moiety) yields compound (B). Glucosamine ingredient The selective deprotection and acetylation of is essentially as described in N1colaou et al. Compound (C) is obtained. (C) and sialyl as mentioned above Reaction with transferase gives the desired product 5LX-R, which It can be produced in relatively low yield using scheme ①.

Including the modified fucoside in the reaction scheme of the synthesis to create SLXs that differ in this component. You can get a likeness. For example, α-D-arabinosyl glycoside is a known Procedure, N1colaou et al., J. Am. Chem. Soc, 112:3693 -3695 (1990), tri-benzyl arabinosyl halide can be used and synthesized. other aryls or alkyls and references therein. References) and can be introduced into trisaccharides in the same way. these Add everything here by quote.

Partial deprotection of trisaccharide (A) according to alternative reaction scheme IIa to produce (D), which was subsequently subjected to cettylation (1991X). chromatograph according to the procedure described in (incorporated herein by reference). After purification with water, (F) is produced.

(F) was sequentially reacted with methylhydrazine, N-acetylation, and O-deacetylation. and ester hybridization yields 5LX-R. .

Preferred examples of Reaction Schemes ■ and IIa include: alkyl ( linear, branched, saturated, monounsaturated and polyunsaturated): Serine (D or L): Serine-containing peptidodi- and tri-alkanolamines (e.g. (H O(CHz), )tNH.

(HO(CH2)-) x N; where n = C2C2+1, linear, branched linear, unsaturated, ■ unsaturated and polyunsaturated I@ sum). R can also be as desired by a person skilled in the art. Use includes aryl, substituted aryl (e.g., Me, OH, T ;”’I alone or in combination), alkylaryl, aryl It can be a component of Lukil or Pond. Phenolic compounds, e.g. The introduction of iodine into syns is known in the art. Contains radical groups Phenols with 12' summer are useful for the introduction of radioactive isotopes and diagnostic produces compounds that are useful in

Compounds comprising SLX and related structures also inhibit selectin-mediated used to measure for the presence of other compounds that can inhibit intercellular adhesion. can be used. Inhibiting selectin-mediated responses using a number of methods Biological activity of test compounds can be assayed for potency. ideally The assay of the present invention can be used to perform large-scale in vitro or in vivo screening of various compounds. This allows for

Reagents or test compounds to be screened are typically synthetic or naturally occurring. The biomolecules produced, e.g. peptides, polypeptides, proteins (e.g. monoclonal antibodies), carbohydrates (e.g. oligoN), glycoconjugates, nucleic acids, etc. Probably. The compounds can be synthesized synthetically using, for example, the methods for synthesizing oligosaccharides described above. produced (also Kbdem, Carbohydrate Chemist ry (Acade+nic Press, Sun Diego, California, 1 988), incorporated herein by reference). of defined composition Methods for synthesizing polypeptides are well known in the art (see At Herton et al., 5solid Phase 5 synthesis (IRL Pre ss, Oxford, 1989), which is incorporated herein by reference). synthesis If the test compound is a polymer (e.g. a polypeptide or polypeptide), it They are preferably modified in a systematic manner to achieve the same sequence of monomers with the desired effect. (see, e.g., U.S. Pat. No. 4,833,092, incorporated herein by reference). Therefore, it is added). The test compound can also be derived from any natural source, e.g., animal, plant, fungal. , or isolated from bacterial cells according to standard procedures described above.

Potentially useful monoclonal antibodies can be obtained according to standard methods described in more detail below. It can be prepared easily.

The assay of the present invention is performed on compounds that act as antagonists or agonists of the ligand molecule. This is useful when identifying antagonistic factors reverse the physiological effects of the ligand or Alternatively, it is a compound that eliminates binding of the ligand to the receptor. The antagonistic factor is It usually competes directly or indirectly with the ligand for receptor binding sites. to reduce the proportion of ligand molecules that bind to the receptor. Typically, the Antifactors are topographical equivalents of natural ligands and directly competes with the ligand for binding sites. Put a compound like this here. It is called rtM (mime tiC) J.

1tisLX (SLXmtimetic) is that it is a selectin receptor. Conformation as an alternative factor for SLX components in recognition by pigs It is a molecule that acts both functionally and functionally. Alternatively, the ligand and test compound Compounds are non-competitive if they can bind to the receptor at the same time. Non Competitive inhibitors do more than reduce the proportion of ligand molecules bound to the receptor. However, it may reduce or inhibit the subsequent physiological effects of receptor-ligand interactions. It works by causing harm. Finally, the measurement method of the present invention can be used to synthesize or a naturally occurring agonist, i.e., binds to a receptor and has a natural ligand Compounds can be identified that initiate a response similar to the physiological response of a human.

Multiple in vitro screening methods for inhibitors of ligand-receptor interactions A number of direct and indirect methods are available and known to those skilled in the art.

For example, inhibiting the attachment of cells with SLX to cells expressing specific selectins can determine the ability to As mentioned above, selectin receptors are This allows the gene to be transferred to cells of a wide variety of species, such as CO8 cells. cells, CHO cells, and the like.

Furthermore, cells that do not normally express SLX synthesize the ligand into transformed cells. ■ or two or more glycosyltransferase genes that confer the ability to Can be transformed. (See, e.g., Lowe et al. Ce1l 63:47 5-484 (1990), which is incorporated herein by reference. ) Typically, Immobilization of test compounds or reagents on cells and solid surfaces with labeled SLX Incubate with activated inversion cells. Then inhibition of cell attachment is determined by detecting the label bound to the surface after appropriate washing. under In the exemplary measurements described in , promyeloid HL-60 cells and activated Using human endothelial cells or activated platelets.

Now that a specific ligand for the selectin receptor has been identified, it is possible to isolate it. Ligand molecules can also be used in measurements. The term “isolated cells” As used herein, "lectin binding factor" or "isolated SLX moiety" other than in its native state, e.g., on the plasma membrane of cells that normally express the ligand. It means a selectin-binding compound that is not linked to the selectin. In this way, the isolated SLX part The component is an isolated molecule, e.g. an oligosaccharide or one component of the molasses itself. Can be done. The isolated molecule can be synthesized or added to the membrane of a cell containing SLX. It can be prepared from Alternatively, isolated selectin-binding factors or S The LX component may be loaded into liposomes or added to the liposomes prior to use in the assay. Can be attached to solid surfaces. Prepare liposomes with selectins Methods and methods of immobilizing agitating biomolecules are described in detail below.

Typically, in vitro measurements of the present invention involve determining whether binding to a receptor or ligand is detect the ability of a test compound to competitively inhibit the binding of a compound known to be , is a competitive measurement. Typically tests for binding between SLX and receptor or ligand do. Inhibition of other binding interactions may also be appropriate, e.g. body (e.g. between FE(6) and SLX or between m5Lx and selectin inhibitor) Inhibition of binding between can be used. Many types of competitive measurements are known (see, e.g., U.S. Pat. No. 3.376,110, U.S. Pat. No. 4.016) ．． No. 043, and Harlow and Lane, Antibodies: A laboratory Manual, Co1d Spring Har bor Publications, New York (1988), here (add by citation).

The assays of the invention also allow for the determination of one component alone, rather than using competitive measurements. It is also suitable for measuring binding of test compounds. For example, using immunoglobulin, SL Compounds containing component X can be identified. for monoclonal antibodies Standard procedures can be used, for example ELISA (see Harlow and Lane, supra). When measuring glycolipid containing SLX antigen , the reactivity between monoclonal antibodies and antigens was originally determined by TLC immunostaining (1983). ) can be measured by solid-phase radioimmunoassay as described in Wear. These documents are included here by citation. Glycoproteins are standard immunoglobulins The rotting procedure was as described by Harlow and Lane, supra. It can be measured in this way. The sandwich assay format also suitable (see, e.g., U.S. Pat. No. 4.642.285 and U.S. Pat. 4.299.916 and U.S. Pat. No. 4.391.904, and Harl ow and Lane, supra, all incorporated herein by reference). typical Specifically, compounds identified in binding assays may be further tested to determine receptor binding. The ability to inhibit Gand interactions can be determined.

Other measurement formats are used to develop ligands or selectins that result from interactions. including detecting the presence or absence of various physiological changes in cells that . Examples of suitable measurements include: binding-induced changes in transcriptional activity. (see, e.g., EPO Publication No. 3712820), various cell media Detection of mediated extracellular effects (see e.g. PCT Publication No. 90100503), and detection of changes in the membrane potential of individual cells (see, e.g., U.S. Pat. ．． No. 343.782), all of which are incorporated herein by reference. or , detect conformational changes in isolated receptors or ligands. See, for example, US Patent i! No. 4.859.609, this It is added here by citation.

Any components of the assay, including the ligand, receptor, or test compound, are placed on a solid surface. Can be bonded to a surface. Numerous methods for immobilizing biological molecules on solid surfaces include: known in the field of For example, a solid surface may be a membrane (e.g., nitrocellulose). ), microtiter plates (e.g. PVC or polystyrene) or It can be beads. Desired components can be covalently or can be attached non-covalently.

A wide variety of natural and synthetic organic and inorganic polymers can be used as solid surface materials. and can be used. Exemplary polymers include: polyester Tyrene, polypropylene, poly(4-methylbutene), polystyrene, polymeth Acrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl) nylbutyrate), silicone, polyformaldehyde, cellulose, cellulose suacetate, nitrocellulose, etc. Other materials that can be used are paper, glass, and paper. Includes lamic, metal, metalloid, semiconductor material, and cermet. moreover , niger-forming substances, including proteins, e.g. Gelatin, ribopolysaccharides, silicates, agarose and polyacrylamide or may contain some aqueous phase-forming polymers, e.g. dextran, polyalkylene glycols (alkylenes of 2 to 3 carbon atoms) or surfactants, e.g. Mediatic compounds, such as phospholipids, long chain (12-24 carbon atoms) alkylammonides Um salt etc. When a solid surface is porous, the various pore sizes can be determined depending on the nature of the system. can be used.

When preparing a surface, several different materials, especially laminates, can be used to properties can be obtained. For example, protein coatings, e.g. gelatin Avoid non-specific binding, simplify covalent conjugation, and Detection can be enhanced, etc.

If covalent bonding between a compound and a surface is desired, the surface is usually polyfunctional or Alternatively, it can be polyfunctionalized. exists on the surface and can be used in chains Functional groups that can be formed include carboxylic acid, aldehyde, amino group, cyano group, ethylene group, It can include hydroxyl groups, mercapto groups, and the like. Wide range of compounds Methods of bonding objects to various surfaces are well known and detailed in the literature. Illustrated. See, e.g., [mmobilized Enzymes, Itir.

Chibata, Halsted Press, New York, 1978, and Cuatrecasas.

J., J. Biol, Chem, 2453059 (1970), this Add this by citation.

In addition to covalent binding, various methods of non-covalently binding assay components are typical. Specifically, it is the non-specific absorption of compounds onto surfaces. Typically, the surface is coated with a second compound. Blocking to prevent non-specific binding of labeled measurement components. Or table Surfaces are designed to nonspecifically bind one component but not significantly bind other components. Measure. For example, surfaces with lectins, e.g. concanavalin A, may contain carbohydrates. with a labeled protein that binds to a compound containing glycosylation but lacks glycosylation. Not combined. Various solid surfaces for non-covalent attachment of measuring components include: U.S. Pat. No. 4.447.576 and U.S. Pat. No. 4.254.082 (which (herein incorporated by reference).

Many assay mats contain labeled assay moieties, e.g. SLX ligand, Using LX, immunoglobulin, receptor, or test compound. The sign is this cup directly or indirectly to the component of measurement according to methods well known in the art. Can be ringed.

A wide variety of labels can be used. Ingredients can be prepared in one of several ways. Can be labeled. The most common method of detection is 2H.

Autoradiation using compounds such as 12″r, 25s, +-c or “P” It's an ophthalmology. The selection of radioisotopes is based on ease of synthesis, various stability depending on the nature of the study, and the half-life of the isotope chosen.

Other non-radioactive labels include ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, Enzymes and act as members of specific binding pairs for labeled ligands This includes antibodies that can be used. The choice of label depends on the required sensitivity and conjugation capacity with the compound. Depends on ease of use, stability requirements, and available instrumentation.

- Non-radioactive labeling is often achieved by indirect means. Generally, the ligand molecule (eg, biotin) is covalently attached to the molecule. The ligand is then an anti-ligand (e.g. streptavidin) molecule, where the antiligand molecule is or a signal system, e.g. a detectable enzyme, a fluorophore or to a chemiluminescent moiety. Ligands and antireactive Gund can vary widely. If the ligand is a natural antiligand, e.g. If it has chin, thyroxine and cortisol and cortisol, it is labeled can be used in combination with natural antiligands. Or Hap Tens or antigenic compounds can be used in combination with antibodies.

Molecules can also be linked to signal-generating compounds, for example by conjugation with enzymes or fluorophores. , can be directly joined. The enzyme in question as a label is primarily hydra enzymes, especially phosphatases, esterases and glycosidases, or oxy- Doreductase, especially peroxidase. Fluorescent compounds are fluorescein Cein and its derivatives, rhodamine and its derivatives, dansyl, umbelif Includes Elon, etc. Chemiluminescent compounds include luciferin and 2,3-dihydrogen. Includes dolphthalazinediones, such as luminol. can be used For an overview of various signal generation systems, see U.S. Patent No. 4.391.904. About Teru. I'll add this here by citation.

As previously mentioned, various SLX units comprising an accessible SLX or 4uSLX In addition to compounds of inhibitors of selectin-mediated cellular Monoclonal antibodies that can inhibit adhesion between Provides a method for identifying.

Monoclonal antibodies bind to selectin ligands or receptors and to block cell attachment. Thus, the production of various immunoglobulin molecules and manipulation techniques available to those skilled in the art to prevent cell-cell adhesion. can cause harm.

As used herein, the term "immunoglobulin" refers to the protein produced by immunoglobulin genes. A protein consisting of one or more qualitatively encoded polypeptides Ru. The immunoglobulin genes recognized are kappa, lambda, alpha, gamma, The delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin Includes the gene for the bulin variable region. Immunoglobulins come in various forms other than antibodies. present in Fv, Fab, and F(ab')t, as well as in -heavy chains. (e.g., Huston et al., Proc. Nat. 1. Ac ad.

Sci. U. S. A. 85:5879-5883 (1988) and Bir. d et al., 5science 242:423-426 (1988)) Add by citation to. (See generally, 1 (ood et al., (mmuno iog y, 2nd edition, Benjamin, New York (1984), and Hun. Kapiller and Hood, Nature 323:15-16 (1 986), which are incorporated herein by reference. ) Antibodies that bind SLX antigens can be generated by a variety of means. More than human The production of monoclonal antibodies in other species, such as rats, lagomorphs, and horses, is well known. and the animal is exposed to the SLX antigen, or a glycoprotein or protein containing the antigen. or glycolipid-containing preparations. immunized animal Immortalize and screen antibody-producing cells obtained from production of antibodies that inhibit the interaction between virus surface proteins and receptor molecules. Screened for life and then immortalized.

For a description of the general procedure for the production of monoclonal antibodies, see t(arlo w and Lane, Antibodies, A Laboratory M anua! (1988), supra.

Human antigens (in the case of SLX units isolated from human tissues) Generation of noclonal antibodies may vary with common techniques. thus, The antigen-binding region of a non-human antibody, such as the F(ab')t or hypervariable region, human constant region (Fc) or framework region using recombinant DNA technology. can be translocated to produce substantially human molecules. This method is common known in the art and, for example, US Pat. No. 4.816.39. 7, European Patent Publication No. 173,494 and European Patent Publication No. 239.400 (these are incorporated herein by reference). Or human SL D encoding a human monoclonal antibody or a portion thereof that specifically binds to The NA sequence was obtained from Huse et al., 5science 248:1275-181 (+9 from human B cells following the general procedure of 89) (which is incorporated herein by reference). DNA library of the desired specificity (or isolated by cloning and amplifying the sequence encoding the binding fragment can do.

A number of currently available monoclonal antibodies can be used in accordance with the present invention to detect selectins. can inhibit cell-cell adhesion mediated by For example, C3LEX- 1 (Reference, (: ampbell et al., J. Biol. Chem.

259:11208-11214 (1984)), slightly different from S L VIM-2 (see Macher et al., supra), FH6 (U.S. No. 4.904.596) (all references cited herein) or Dr. S. Hkomo (Biomenbra) neInstitute, Seattle, Washington), and is SH,.

Compounds of the invention, including immunoglobulins, can be used in the preparation of pharmaceutical formulations as described below. It can be used at any time. When the compound is an oligosaccharide or sugar liquid combination, S Although the LX or pseudo-SLX moiety can be provided in a variety of forms, selectin receptors Sebutar, e.g. with ELAM-1, GMP-1,40 or MEL-14 antigens It should be able to bind effectively and thereby inhibit cell-cell adhesion.

The pharmaceutical compositions of the present invention block cell adhesion or can be inhibited. For example, many inflammatory diseases affect vascular endothelial cells and platelets. related to selectins expressed on

The term ``inflammation'' is used here to refer to both specific and non-specific IW reactions. means. A specific defense system response is a specific immune system response to an antigen. singular Examples of protective system reactions include antibody responses to antigens, such as viruses, and delayed hypersensitivity. Encompasses agility. Non-specific defense system responses are generally associated with white blood cells for which immunological memory is not possible. It is an inflammatory response mediated by blood cells. Such cells are macrophages, Includes acidophils and neutrophils. Examples of non-specific reactions are immediate swelling after a bee sting; Collection of PMN leukocytes at the site of bacterial infection (e.g., in bacterial pneumonia) pulmonary infiltrates and pus formation in abscesses).

Other treatable diseases include, for example, rheumatoid arthritis, ischemia, Leukocyte-mediated tissue damage after (reperfusion injury), frostbite injury or shock, acute leukemia bulbar-mediated lung injury (e.g. respiratory distress 11ffl syndrome in adults), asthma, traumatic events. shock, septic shock, nephritis, and acute and chronic inflammation, e.g. peptic dermatitis, psoriasis, and inflammatory intestinal diseases. Various platelet-mediated pathological events For example, allelic arteriosclerosis and coagulation can also be treated.

Furthermore, tumor metastasis can be inhibited or prevented by inhibiting the adhesion of circulating cancer cells. I can do it. Examples include colon cancer and melanoma.

As an example, reperfusion injuries are particularly amenable to treatment with the compositions of the present invention. GM Compositions that inhibit P-140 selectin-ligand interaction are particularly useful for reperfusion. May be useful in treating or preventing injuries. The present invention can be used as a preventive measure before cardiac surgery. It can be used to enhance recovery after surgery.

GMP-140 is a Weibel conjugate of platelets and endothelial cells. -Palade) after being stored in the body and activated by thrombin GMP-140-ligan, as it is released and mediates the adhesion of neutrophils and monocytes. Inhibitors of this interaction minimize the tissue damage that often accompanies thrombotic diseases. It may be particularly useful in some cases. For example, such inhibitors may , especially in patients who have experienced myocardial infarction, deep vein thrombosis, pulmonary embolism, etc. Has therapeutic value. The compounds are particularly useful in thrombolytic therapy.

The compositions of the invention can be used to treat patients secondary to septic shock or disseminated intraductal coagulation (DrC). Particularly used to treat effects. Groups during septic shock or DIC Migration of leukocytes into the tissue often results in pathological tissue destruction. Furthermore, this These patients may have widespread microcirculatory thrombosis and diffuse inflammation. Here The pharmaceutical compositions provided in this article inhibit leukocyte migration in these areas and to alleviate tissue damage.

Inhibitors of selectin-ligand interactions also play a role in traumatic shock and its effects. It is useful in the treatment of acute tissue damage associated with this. Selectins are involved in damage Replenishment of leukocytes to the body, especially in the case of active injury and inflammation, and recruitment of ELAM-1 These inhibitors can be administered locally or systemically to Tissue damage associated with such injuries can be inhibited. Moreover, inflammation Due to the site specificity of such inhibitors, e.g. When compared to traditional anti-inflammatory agents, these compositions may be more effective. Yes, and less likely to cause complications.

Thus, the present invention also provides pharmaceutical compositions that can be used in treating the aforementioned conditions. provide The pharmaceutical composition may contain biomolecules or other compounds comprising SLX units. , an antibody that binds to SLX, or between an SLX ligand and a selectin receptor other compounds that inhibit the interaction of the drug, and a pharmaceutically effective carrier. There is.

The biomolecules of the invention include peptides, polypeptides, proteins (e.g., immunoglobulins). (brine), carbohydrates (e.g. oligosaccharides or polysaccharides), sugar complexes (e.g. glycolipids or glycoproteins), nucleic acids, etc. pharmaceutical composition are suitable for use in a variety of drug delivery systems. Simple method of the present invention for delivering drugs l! Regarding the view, see Langer, 5science 249: 1527- 1533 (1990). I'll add this here by citation.

In light of the complexity of inflammatory responses in mammals, those skilled in the art will readily appreciate that In addition, the pharmaceutical composition of the present invention combines a compound that promotes SLX and functions of other cell adhesion molecules. It can be mixed with other compounds known to interfere. for example , a member of the integrin family of adhesion molecules, is involved in leukocyte extravasation at the point of infection. considered to play a role. Intercellular adhesion receptors, including selectin receptors For an overview of proteins and their role in immune function, see Sprlnger.

See Nature 346: 425-434 (1990). this Added here by citation. Furthermore, methods for successfully using the pharmaceutical compositions of the present invention The location can also be determined by the state of development of the condition to be treated.

Different adhesion molecules play a role in response to various factors during the course of a disease or condition. As one skilled in the art will recognize, different pharmaceutical compositions can be adjusted up or down. may be required for the treatment of different inflammatory conditions.

In one embodiment, the SLX ligand of the pharmaceutical composition is used to provide common anti-inflammatory properties. Drugs or other agents can be focused to specific sites of tissue damage. Sele Cutin-binding components, such as SLX ligands or pseudo-SLX, can be used to sequester drugs. Concentrates on lectin receptors, e.g. selectin receptors on vascular endothelial cells. By allowing such drugs to achieve higher concentrations at the site of injury, can be done. Side effects from common anti-inflammatory chemotherapeutics can be reduced by lower doses, Localization of the drug at the site of injury and/or encapsulation of the drug prior to delivery may result in substantial relief. can be reduced.

Targeting component, i.e. SLX ligand or desired selective Pseudo-SLX that binds to tin can be coupled directly or indirectly to chemotherapeutic agents. I can do it. It can be carried out by means generally known in the field. A coupling that substantially inhibits the ability of the ligand to bind to the receptor. Or Chemistry 1! It should not substantially reduce the activity of the drug. seed Various chemotherapeutic agents can be coupled for targeting. example For example, chemotherapeutic agents that can be coupled include two-pronged agents including: Compounds with bright SLX, immunomodulators, platelet activating factor (PAF) antagonists gonists, cyclooxygenase inhibitors, lipoxygenase inhibitors, and Leukotriene antagonist. Some preferred ingredients include cyclosporine A , indomethacin, naproxen, PK-506, mycophenolic acid, etc. Contains. Similarly, antioxidants such as superoxide dimutase, SL Treatment of reperfusion injury when targeting with X-ligand or pseudo-SLX It is useful in Similarly, anticancer drugs use SLX ligands or pseudo-SLX as chemotherapeutics. Targeting can be achieved by coupling to a therapeutic agent. Cool Examples of factors that can be pulled are dauromycin, doxorubicin, vin Includes blastin, pleomycin, etc.

Targeting of selectin receptors can also be achieved by targeting them as aggregates in aqueous solution. Amphiphiles or molecules of dual character (polar.

(non-polar). 100H medium substances include non-polar lipids and polar lipids. sugar, monosaccharides and trisaccharides, sulfatides, lysolecithins, phospholipids, saponins, bile Includes acids and salts. These molecules form emulsions and foams, micelles, and insoluble monomers. They can exist as layers, liquid crystals, phospholipid dispersions and lamellar layers. this are commonly referred to herein as liposomes. These gIiI! In things, supplies The drug to be delivered may be SLX ligand or selectin as part of the liposome. It is incorporated in combination with sLX, which binds to the receptor.

In this way, liposomes loaded with the desired chemotherapeutic agent are placed at the desired site of tissue damage. can be directed by lectin-3LX ligand interaction. The liposome When delivered to the vicinity of affected cells, liposomes deliver the therapeutic composition of choice. supply.

The liposomes of the invention are formed from standard vesicle-forming lipids; Neutral and negatively charged phospholipids and sterols, e.g. cholesterol includes. Lipid selection generally depends on, for example, liposome size and blood flow. It is determined by considering the stability of the liposomes inside.

Typically, the major lipid component within liposomes is phosphatidylcholine.

Phosphatidylcholines with various acyl chain groups of various chain lengths and degrees of saturation is available or can be isolated or synthesized by well-known techniques. Can be done. In general, phosphatidylcholine with low saturation is used for filter sterilization purposes. , especially when the size of liposomes must be reduced to approximately 0.3 microns or less. , the size can be determined more easily. Determination of liposome size and filtration The method used for bacteria is described below. The composition of the acyl chains of phospholipids also may affect the stability of the liposomes inside. One preferred phosphatide Zircholine is partially hydrogenated egg phosphatidylcholine.

Various targeting agents (e.g., ligands, receptors and monochromes) Targeting of liposomes using neutral antibodies is well known in the field. It is being (See, e.g., U.S. Pat. No. 4.957.773 and U.S. Pat. No. 4.603.044, both of which are incorporated herein by reference). various minutes Using small amounts of glycoproteins and glycolipids as targeting agents be able to.

Typically less than or equal to about 300,000 Daltons, preferably from about 40,000 to about 2 50,000 Daltons, more preferably about 57,000 to about 150,000 Daltons Uses Tong protein that grows molecular weight of tons. Approximately 10,000 Daltons or less, Preferably, a glycolipid with a molecular weight of about 600 to about 4,000 Daltons is used. .

Using exploratory methods to couple targeting agents to liposomes I can do it. These methods generally involve targeting liquid components, e.g. Phosphatidylethanolamide that can be activated for factor attachment or derivatized lipophilic compounds, e.g. lipid derivatized pleomycin. Incorporating the drug into liposomes. Antibody targeted liposome The membrane can be constructed using, for example, liposomes incorporating protein A. (see Renneisen et al., J. Biol. Chem. , 26516337-16342 (1990) and Leonetti et. at, Proc, Natl, Acad, Sci.

Generally speaking, the targeting mechanism is that the targeting factor is a selectin. The targeting factor in such a manner that it is available for interaction with the receptor. on the surface of the liposome. Liposomes are typically , configured in such a way that a portion of the connector is first incorporated into the membrane during membrane formation. be done. The connector portion is oleophilic, firmly embedded and anchored within the membrane. Must have a part. It is also possible to chemically It must have a hydrophilic moiety that is available for use. Hydrophilic part added later chemically suitable to form a stable chemical bond with the targeted targeting agent. As it is, it is selected.

Therefore, the connector molecule reacts with the targeting agent and has a hydrophilic reaction. to hold the functional group in its correct position and extending out from the surface of the liposome. It must have both suitable lipophilic and hydrophilic reactive groups for this purpose. be In some cases, the targeting agent can be attached directly to the connector molecule. However, in most cases this is accomplished using a third molecule that acts as a chemical bridge. In this way, the connector molecules present in the membrane are transferred from the inside of the vesicle surface to the outside in a three-dimensional manner. It is even more appropriate to link with extended target factors.

The charge of liposomes is an important determining factor when clearing liposomes from the blood. The negatively charged liposomes are taken up more rapidly by the reticular endothelial system ( Juliano, Biochem, Biophys, Res, Comm.

63:651(1,975)), thus having a shorter half-life in the bloodstream Ru. Liposomes with extended circulating half-lives are typically used for therapeutic and diagnostic purposes. desirable for use. Limestone that can remain in the bloodstream for up to 8, 12 or 24 hours Posomes provide sustained release of selectin-ligand inhibitors of the invention or the inhibitory factor (which can be labeled and target the desired area (can provide diagnostic images within the body) This can be promoted.

Typically, about 5-15 mol% of negatively charged phospholipids, e.g. Contains ruglycerol, phosphatidylserine or phosphatidylinositol Prepare liposomes. Added negatively charged phospholipids, e.g. Tidylglycerol prevents spontaneous liposome aggregation and thus eliminates unwanted liposomes. The risk of formation of soma aggregates can be minimized. Membrane stiffening factors, e.g. Pingomyelin or a saturated neutral phospholipid (at a concentration of at least about 50 mol%) ) and 5 to 15 mole % monosialyl gangliosides, generally described in U.S. Pat. No. 4.837.028 (which is incorporated herein by reference). Thus, circulation of liposome preparations in the bloodstream can be increased.

Additionally, liposome suspensions are susceptible to free radical and lipid peroxidation during storage. May contain lipid protective factors that protect liposomes and drugs against damage . Quenchers of lipophilic free radicals - 1 e.g. alpha-tocopherol and water-soluble Iron-specific chelators, such as ferriocyanine, are preferred.

For example, 5zoka et al., Ann, Rev. Biophys, Bioeng, 9 : 467 (1980), U.S. Patent No. 4.235.871, U.S. Patent No. 4. No. 501.728 and U.S. Pat. No. 4.837.028, each of which is incorporated herein by reference. Various methods of preparing liposomes are available, as described in Available.

One method produces multilamellar vesicles of non-uniform size. In this method, small The vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dissolved under vacuum or Dry under inert gas to form a thin lipid film. If necessary, use this The film is dissolved in a suitable solvent such as tert-butanol and then lyophilized. to form a more homogeneous liquid mixture, which is a powder-like form that hydrates more easily. It is a state. This film targets the drug and the targeting ingredient in water. Cover with solution and allow to hydrate, typically with stirring for 15-50 minutes.

The size distribution of the resulting multilamellar vesicles is significantly different under conditions of more intense selectin receptors. By hydrating the lipids or adding solubilizing detergents, smaller It can be shifted to a larger size.

The hydration medium is at the desired concentration in the internal volume within the final liposome suspension; Contains targeted drugs. Typically, the drug solution is 10-100 mg/m 1 in a buffered physiological saline solution. Targeting component X molecule or The concentration of pseudo-SLX that binds selectins is generally about 0.1-20 mg/ml. be.

After liposome preparation, the size of the liposomes can be regulated to achieve the desired size range and size. and a relatively narrow distribution of liposome sizes can be achieved. one preference The size range is about 0.2 to 0.4 microns, which is similar to ordinary filters, The liposomes are typically filtered through a 0.22 micron filter. Allows for sterilization of the suspension of somes. The filter sterilization method uses approximately 0 liposomes. ．． If the drop is between 2 and 0.4 microns, it can be carried out on a high production basis. Ru.

Several techniques are available for sizing liposomes to the desired size.

One sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. ). Liposomes by bath or probe sonication Sonication of the suspension produces small monolayers of size smaller than about O,O5 microns. resulting in a gradual size reduction into vesicles. Homogenization reduces large liposomes to smaller Another method relies on shear energy to fragment particles. Typical homogenization hand In order, the multilamellar vesicles are recirculated through a standard emulsion homogenizer for selection. selected size, typically about 0.1 to about 0.5 microns. . In both methods, the particle size distribution is similar to that of a normal laser beam. can be monitored by discrimination.

Liposoring through porous polycarbonate membranes or asymmetric ceramic membranes Extrusion of the liposome also reduces the size of the liposomes into a relatively well-defined size distribution. This is an effective way to reduce Typically, the suspension is adjusted to the desired liposome size. Cycle through the membrane one or more times until distribution is achieved. Liposomes are Gradually limit liposome size by extruding through successively smaller pore membranes do.

Even with the most efficient encapsulation, initial liposome suspensions contain free (unentrapped) 50% or more of the drug and targeting agent May contain. Therefore, free drug or targeting agent is It is important to remove the final injectable suspension.

Several methods can be used to remove unentrapped compounds from liposome suspensions. can be used. In one method, liposomes in suspension are Free compounds and very small liposomes are removed from the supernatant by precipitation. leave it inside. Another method involves concentrating the suspension by ultrafiltration and then This involves resuspending the somes in a drug-free displacement medium. Or gel Filtration can be used to separate large liposome particles from solution molecules.

After treatment to remove free drug and/or targeting factor, the liposome The suspension is brought to the desired concentration for intravenous administration.

This can be done if the liposomes are concentrated, e.g. by centrifugation or ultrafiltration. Alternatively, if the drug removal step increases the total suspension volume, By concentrating, liposomes can be resuspended in the injection medium. . The suspension is then sterilized by filtration as described above. liposome-ligand The preparations can be administered parenterally or topically, where the dosages are, for example, For example, it will vary depending on the method of administration, the drug to be delivered, the particular disease being treated, etc.

Contains SLX ligand and/or pseudo-SLX that binds to selectin receptor. For pharmaceutical compositions consisting of the method, the specific disease being treated and its severity, the patient's overall health and condition, and and the prescribing physician. For example, for the treatment of reperfusion injuries, Dosage ranges from approximately 50 μg to 2,000 mg/day for a 70 kg patient . Ideally, treatment administration should begin as soon as possible after myocardial infarction or pond injury. It is possible. Pharmaceutical compositions can be used for parenteral, topical, oral or topical administration. , e.g. by aerosol or transdermally, for prophylactic and/or therapeutic treatment. intended for placement. Pharmaceutical compositions can be prepared in a variety of unit dosage forms depending on the method of administration. It can be administered in

For example, unit dosage forms suitable for oral administration include powders, tablets, pills, capsules and and sugar preparations.

Preferably, the pharmaceutical composition is administered intravenously. Thus, the present invention can be used as a medicine. A solution of the compound dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A composition for intravenous administration is provided. Various aqueous carriers, such as water, Buffered water, 0.4% saline, etc. can be used. The composition of these Items should be sterilized by normal, well-known sterilization techniques or filter sterilized. be able to. The resulting aqueous solution may be packaged as is or freeze-dried. The lyophilized preparation is combined with a sterile aqueous solution prior to administration. Composition is a pharmaceutically acceptable auxiliary side, as required for the approximate physiological condition, e.g. For example, pH adjusting agents and buffering agents, tonicity adjusting agents, wetting agents, etc., such as sodium acetate. um, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sol Contains bitan monolaurate, teriethanolamine oleate, etc. can.

with other SLX ligands or pseudo-SLX to increase efficacy in pharmaceutical formulations. SLX ligands or pseudo-S that can be combined to form “cocktails” The concentration of LX can vary widely, for example from concentrations below about 0.05%, usually at least It can vary from 1% by weight to as high as lθ~30% by weight, and mainly The selected mode will be determined according to the fluid volume, viscosity, etc. cormorant. The cocktail also includes monoclonal antibodies that bind to selectin receptors, e.g. For example, monoclonal antibodies against ELAM-1 or GMP-140 can be In combination with X-ligand, pseudo-ligand or monoclonal antibodies against the ligand In addition, the ligand-receptor interaction can be efficiently inhibited. . As previously mentioned, the components of the cocktail can be delivered via liposomal preparation. Wear.

Thus, a typical pharmaceutical composition for intravenous infusion consists of 250 ml of sterile Ringer's solution, and can be configured to contain 25 mg of the compound . Are practical methods of preparing parenterally administrable compounds known to those skilled in the art? , or obvious and, for example, Remington's Pharm aceutical 5ceinces, 17th edition, MackPublish r+g Co [l1pany, Easton, Pennsylvania (1985) (this (incorporated here by reference).

For solid compositions, common non-toxic solid carriers can be used; For example, pharmaceutical grade mannitol, lactose, starch, stearate Gnesium, sodium saccharin, talc, cellulose, glucose, sucro carbonate, magnesium carbonate, etc. Pharmaceutically acceptable for oral administration The non-toxic composition may contain excipients normally used, such as any of those listed above. and generally 10 to 95% of the active ingredients, i.e. one or two of the invention. By incorporating more than 20% of the SLX ligand or pseudo-SLX, preferably 20% (Remington"S, supra).

For aerosol administration, the compound is preferably in finely divided form with a surfactant or and propellant. SLX oligosaccharide ligand or pseudoligand Typical percentages are 0.05-30% by weight, preferably 1-10% by weight. world The surfactant is, of course, non-toxic and preferably soluble in the propellant. this Representative of such substances are fatty acids containing 6 to 22 carbon atoms, e.g. , caproic acid, octanoic acid, valmitic acid, stearic acid, linoleic acid, linoleic acid acids, orestearic acid and oleic acid and aliphatic polyhydric alcohols or their cyclic Anhydrides such as ethylene glycol, glycerol, erythritol, arabic Toll, mannitol, sorbitol, hexyto derived from sorbitol polyoxyethylene and polyoxypropylene anhydrides, and their esters. Esters or partial esters with pyrene derivatives. mixed esters, e.g. , mixed or natural glycerides can be used. The surfactant is the component of the composition. It constitutes 0.1-20% by weight, preferably 0.25-5% by weight. rest of the composition is usually a propellant. Liquefied propellants are typically gaseous at ambient conditions. , and is condensed under pressure.

Examples of suitable liquefied propellants are lower alkanes containing up to 5 carbons, e.g. Butane and propane, preferably fluorinated or fluorinated alkanes. Ru. Mixtures of the above can also be used.

When preparing an aerosol, place the finely ground compound and and a suitable propellant containing a surfactant.

The components are thus maintained under high pressure until released by the action of the valve.

Compositions containing the compounds may be administered for prophylactic and/or therapeutic treatment. Ru. In therapeutic applications, the compositions may be applied to patients already afflicted with a disease, as described above. to cure or at least partially cure the symptoms of the disease and its complications. Administered in an amount sufficient to prevent. To achieve this, the appropriate amount is 'therapeutic'. is defined as the effective dose J. An effective amount for this use depends on the severity of the disease. and depending on the patient's weight and general condition, but typically for a 70 kg patient. ranging from about 0.5 mg to about 2.000 mg SLX oligosaccharide or pseudo-SLX/day. and a 7-day dose of about 5a+g to about 200+ng of compound is commonly used.

In prophylactic applications, compositions containing compounds of the invention may be used to treat patients susceptible to a particular disease. administered to patients who have or are otherwise at risk for the disease. this Such an amount is defined as a "prophylactically effective amount." In this use, the exact amount again depending on the health condition and weight of the patient, but generally from about 0.5 mg to about 1.5 mg. 000mg/70kg patients, usually about 5mg to about 200mg/70kg Weight range.

Single or multiple administrations of the composition may be carried out, and the level and pattern of administration may vary. The selection is made by the treating physician. In either case, the pharmaceutical formulation SLX oligosaccharides or pseudo-SLX should be provided in sufficient quantities to effectively treat patients. It is possible.

Compounds of the invention can also be used as diagnostic reagents.

For example, in patients suspected of developing inflammation using unknown compounds. Areas of inflammation or tumor metastasis can be located.

For this use, the compound 1111. +4 (or cleanse with tritium) I can do it.

The invention is further illustrated by the following examples. These examples do not limit the invention. do not.

LECI 1. HL-60, HT-29, certain adenocarcinomas (especially colo20 5 cells) and polymorphonuclear leukocytes (PMNs, neutrophils) contain highly specific fucocysts. Contains transferase I, which sialylates fucose from GDP-fucose. Substrate NeuAcα2゜3Galβl, 4GIcNAc or NeuGca 2,3Galβ1.4G]cNAc.

As is well known in the field, fucose is present in the lumen of the Golgi apparatus. The oligosaccharide chain is transferred via a specific fucosyltransferase, and this is transferred to the 5ch acter and Roseman, rTheBiOChemiStr’10 f　Glycoproteins and Proteoglycans'', W, Lennarzl! , PlenumPress, New York, pp. 85-160 (1980) (which is included here by citation). Ru. The subcellular localization location of fucosyltransferase is within the Golgi apparatus. The first step in isolating these enzymes was to isolate this new and specific α1. 3-F The goal is to isolate the fraction of the Golgi apparatus that expresses cosyltransferases.

The vesicle fraction derived from the Golgi apparatus was analyzed by Balch et al., Ce1l 39:405 ( +984) (which is added here by citation) in a variation of the procedure described in Prepare from LEC11, HL-60, HT-29, PMN.

co1o205 or other containing α1,3-fucosyltransferase I The cell lines are grown in suspension to a density of approximately 5XIO' cells/1. suspend cells 2. From a cloudy culture. Harvested by centrifugation at OOOxg. 12 liters of suspension The precipitate resulting from the solution (6X10”) was treated with Tris-CI (IO mmol), p H7,0, heat-inactivated fetal calf serum (7%) and aprotinin (10%) 0μg/a+l, Siga+a Chemical Co, Missouri, St. Lewis (W/V).

Cells are tightly fitted into Wheaton glass dounces. ce) Crush with a homogenizer using mortar A (60 strokes). Ho Centrifuge the modinate in a tabletop clinical centrifuge at soo xg for 5 minutes. centrifuge Discard any remaining lipids and insoluble material at the top of the solution. cloudy skim Transfer the liquid to a clean tube and then adjust the sucrose concentration of the supernatant using Tris-CI (2 0mM), 40% (W/V) sucrose in pi (7,0) with the aid of a reflectometer. Adjust more. Transfer 5 ml of this suspension to an ultracentrifuge tube and sequentially 2.5 ml 35% (w/v) of Tris-CI (10[11M, pH 7,0)] sucrose and 39% (w/v) in 2.0 ml 1Od Tris-CI buffer ) layer with sucrose. This gradient is 5W-410-tar (Beckman ) for 1 hour at 110.000×g at 5°C. rich in Golgi apparatus Vesicles are collected from an internal phase of 29% to 35% sucrose. other subcellular minutes fractions are found in other internal phases within the gradient; for example, vesicles are Golgi-induced small It is derived from the rough and smooth endoplasmic reticulum bands beneath the follicles. 29%～ The band removed from the 35% internal phase indicates the presence of sialyltransferase activity. and analyze for quantity.

The enzyme sialyltransferase is found within Golgi-induced vesicles. is known and collected from 29-35% internal phase by a person skilled in the art. is used as a marker to assess the truthfulness of sialyltransf The chelase assay was performed by Brles et al., J. Biol. Chell, 25. 2 + 1107 (1977), as an acceptor. Performed using asialofetuin. Excellent Golgi apparatus from LEC cells Preparations of induced vesicles develop specific activities of 3.0 nmoles/mg protein/hour. .

The resulting preparation of the Golgi apparatus is then used in the synthesis of the aforementioned enzymes. ．． Used as a source of 3-fucosyltransferase I.

LEC11 cells (SL) bind to activated endothelial cells expressing ELAM-1. The ability to express X), the structure Le! That is, expressing the non-sialylated form of SLX. compared with that of CHO cells and other glycosylation mutants LEC12.

material Subculture culture grown on gelatin-coated 48-well assay plates. Human venous endothelial cells (HUVECXCl onetics) of 5. used as a source. Cells were diluted with TL-Iβ (Genzyme) to 30 μg/ml. It was stimulating. Cells were stimulated for exactly 4 hours. american type culture ・Collection (American Type C1ture Co11ec tion) (ATCC No., CCL240) in cells with a control ligand. was used as a source. These are penicillin (100 units/ml)/strepto Mycin (100μg/+n1XIrvinScientifc), L-glue Tamin (2mM) (Irvin 5cientifc) and 10% fetal bovine RPMII640 containing blood N (Hazleton) (hereinafter referred to as CRPMT) ) were harvested from bulk cultures. 1 for LEC11, LECI2 and CHO- , P., provided by Dr. Tanley. They are ribonucleotides and and deoxyribonucleotides (Gibco), penicillin (100 units/ml) /Streptomycin (100 units/ml) ([rvin 5cientifc ), L-glutamine (2mM) (Irvin 5cientifc) 10% in suspension cultures in alpha MEM containing calf serum (HaZleton). and multiplied.

procedure 1,, LECI 1. Obtain 1 in LECI2 and CHO-, and CRPM Washed in I. Viable cell counts were performed using Trivan blue. 3 of each type ×lO cells were precipitated in a 10 ml test tube, and 300 μm of “Cr” (450 μCi) (New England Nuclear) was added to each precipitate. added. Incubate the tube at 37°C for 1 hour with gentle agitation. It was done.

2. Wash labeled cells 3x in medium and wash 2x 10'/400μm (6 ml). The test tubes were then placed in a 4°C water bath.

3. After 4 hours of incubation with IL-1β, activated HUVECs were Remove the containing assay plate from the incubator and The samples were cooled for 15 minutes by placing them in a 4°C water bath.

4. When the temperatures in both samples reach equilibrium, remove the medium from the measurement well. Remove several wells at a time with a pipette.

5. Add labeled cells to wells in a volume of 400 μl equal to 2X10' cells/well Added with. Place three 400 μm aliquots of each cell suspension into glass tubes. It was entered to determine the output CPM.

6. The plate was incubated in an ice water bath for 30 minutes.

7. Remove unbound cells from the wells of the assay plate using a Pasteur pipette. Systematic resuspension and subsequent removal (by addition and removal of 0.7 ml of medium) I left.

8. Remove all of the medium from the wells and add 0.125M Tris, 2% A solution of SDS and 10% glycerin was added (0.3 ml).

The plate was left for 30 minutes and then 0.5ml of dH20 was added to each well. .

9. Resuspend the fluid in each well with a P100 pipette and pipette into the glass tube. Transferred to test tube. The tip of PlooO was placed into the test tube.

Count the test tubes containing the IO1 input CPM sample with a gamma counter. I counted it.

11. Divide the bound CPM in each well by the input CPM for each sample. , the % bound was determined. Produce the mean and standard deviation of points for three replicate assays. I hit it.

The results obtained in this experiment are shown in Table 1, which shows that SLX cells that bind effectively to activated vascular endothelial cells expressing ELAM-1. Develop abilities. These data indicate that high levels of unique carbohydrate S LECII cells expressing LX exceptionally bind to IL-1β-activated HUVECs. LEC12 and CHO-Kl that are combined or lack a significant amount of this carbohydrate are It is a poor binder of activated HUVEC. This conclusion further The observation that ELAM-1-mediated binding occurs at 4°C demonstrates the characteristics of ELAM-1-mediated binding. will be supported.

Example■ Inhibition of cell-cell adhesion by monoclonal antibodies specific for SLX The ligand for ELAM-1 on neutrophils is that the terminal sugar is NeuA, cα2, 3Galβ1.4 (α1..3Fuc)GlcNAc(SLX), Two sets of experiments are described that demonstrate the presence of gosaccharide.

These experiments demonstrated that ELA of HL-60 cells to IL-1β-stimulated HUVEC to sialylated Le” and non-sialylated forms of Le, blocking M-1-mediated adhesion. carried out by assaying the ability of monoclonal antibodies specific for .

HUVECs at passage 3 from cultures started for experiments of the present invention were previously described. I used it like this. Two sets of triplicate wells served as controls. four threes Repeat test samples were treated with TL-1β (Genzyme) at 10 μg/ml and Stimulation was performed four times at 20 μg/ml. American type culture collection HL-60 cells obtained from the . These include penicillin (100 units/1 liter), streptomycin (100 μg/ml) (irvin 5cientifc), L-glutamine (2mM) (Irvin 5cientifc) and 10% fetal bovine serum (Hazle ton) in RPM11640 (hereinafter referred to as cRPMI). Obtained from nutrition.

The monoclonal antibody preparation contains approximately 20 μg/ml of 5NH3 (IgM) and Contained SHI (1gG3) at approximately 10μg/ml. 5NH3 is for SLX SHI recognizes non-sialylated structures.

procedure 1. HL-60 was harvested and washed in CRPMI. Count viable cells. This was done using Liban Blue. 3XIO' cells in 2xlOml tube and 300 μm of “Cr (450 μCi) (New English d Nucl, ear) was added to each test tube. Do not stir the test tube gently. and incubated for 1 hour at 37°C.

2. Antibodies were supplied as hybridoma culture supernatants and 0.01% N aNz and 0.05% thimerosal. To remove these preservatives 5111 to each of 3 x 500 ml of legacy tissue culture medium. and dialyzed for 72 hours.

3. Collect antibodies from dialysis and put 3.5ml each into 10ml test tubes . the remainder for use in an ELISA assay for HL-60 binding. was held at Add 7 ml of RPMI 16.5% Fe2 to the fourth tube as a control. I put it in for use.

4. Wash labeled HL-60 three times in CRPMI and place into one tube. I pooled. They were then centrifuged and resuspended in 1 ml in medium. .

5. Add a 200 μm cell suspension to each tube containing the antibody, and 400 μm was added to control tubes. Stir the tube gently until it reaches 37°C. and incubated for 20 minutes.

6. Remove the stimulated HUVEC assay plate from the incubator. , and remove the medium from the wells with a Pasteur pipette, several wells at a time. Ta.

7. 0.5 ml of cell suspension was added to each well in triplicate. contrast at both IL-1β concentrations on unstimulated and stimulated HUVEC cells. I plated it. Test cells were added only to stimulated wells.

8. Add 0.5 ml of the final coat of each cell to the glass tube and adjust the input CPM. was used to determine.

9. Place the assay plate in the incubator (5% CO2, 37”C) for 30 minutes. I set it back a minute.

10. Mix the coat of each cell suspension with an equal volume of trypan blue and Cells were examined for viability microscopically.

The results were as follows: Control = 98%, 5HI = 92%, and 5NH3 = 9 9%.

11. Remove unbound cells from the wells of the assay plate using a Pasteur pipette. by systematic resuspension and subsequent addition and removal of <0.7 ml of medium. Removed.

12. Remove all of the medium from the wells and add 0.125M l-Lis, 2% of SDS (Bi o-Ra d) and 10% glycerin (Fi 5h er) was added (0.3 ml). Leave the plate for 30 minutes and then 16 ml of dH20 was added to each well.

13. Resuspend the fluid in each well with a P100 pipette and pipette Transferred to a test tube. The tip of PlooO was placed into the test tube.

14. Input count 7 minutes (CPM) Gun the test tube containing the sample. I counted it with a macounter.

15. Calculate the bound CPM in each well as the input CPM for each sample. The % combined was determined by dividing. Mean and standard deviation of points from three replicate assays was plotted.

Repetitive experiments are best in experiments where high IL-1β was used to induce endothelial cells. It was determined that

The results showed that monoclonal antibody 5NH3 was able to induce IL- was shown to block the binding of HL-60 cells to 1β-stimulated HUVEC. S The control antibody SHI, which does not bind to the LX determinant, was used to bind HL-60 cells to ELAM-1. did not block the join. This suggests that the terminal scissors in the ligand Alic acid is required for binding to ELAM-1.

B, Monoclonal antibody panel 2 48-well assay plate coated with Seratin (C.

Passage 3 HUVECs grown on 5 tar) were used as the endothelial cell source. . Plates were prepared as described above. Control wells of 3 replicates of 2ffl It was left unstimulated. Seven triplicate specimens on each plate were measured at 0.5[ 0177) E G M - Stimulated with IL-1β in MV at 30 μg/ml did. Cells were stimulated for exactly 4 hours. HL-60, Iff cell (ATCC) was used as a source of cells containing

These were obtained from bulk cultures in CRPMI. Containing monoclonal antibodies The fresh hybridoma supernatant contained: FH6 (IgM). Lower affinity mAb; 5NH-3 (IgMX20Jig/n+1): SH-1 (IgGs) (10 μg/ml); FH-2 (JgM), Le” Reactive mAb: 5NH-4 (IgG3) High affinity antibody: and C3LE X-1 (IgMXP, provided by Dr. Terasaki, UCLA, purified The immunoglobulin used in this assay was 2.8 mg/ml. in Dulbecco's Modified Eagle's Medium (DMEM) containing 5% Fe2 to (diluted to 9 μg/ml). The specific activity of the antibody was as follows.

FH6, 5NH-4, 5NH3 and C3LEX-1 are specific for SLX. FH2 and SHI were specific for non-sialylated Le''.

procedure 1.8L-60 was obtained and washed in CRPMI. Count viable cells. This was done using Liban Blue. Place 3X10@cells into 2XIOml test tube. and 300 μm of “Cr (450 μCi) (New England Nuclear) was added to each test tube. Stir the test tube gently.3 Incubation was for 1 hour at 7°C.

2, [Identification, -HL-60 in DMEM containing 596 (DFC3'c (hereinafter referred to as c DMEM) and pooled into one tube. then , centrifuge them 4 and resuspend them at 4X10' cells/1 ml in the same medium. Ta.

3. 3.2 ml of culture supernatant of each monoclonal antibody, and 3.2 ml of culture supernatant of each monoclonal antibody. ffjlled C5LEX-1 (29 μg) was added to separate tubes; 6.4 ml of medium was added to the test tube.

4. Monoclonalize the 200 μm cell suspension (equivalent to approximately 8 x 10’ JI cells) 400 μm was added to the tube containing the antibody and 400 μm was added to the control tube. tube The mixture was incubated for 20 minutes at 37°C with gentle agitation.

5. Remove the stimulated HUVEC assay plate from the incubator. The wells were then washed once with cDMEM and the medium removed from the wells using a Pasteur pipe. PET was removed, several wells at a time.

6. Added 0.4 ml of cell suspension to each well of one of the two plates.

Both IL-1β concentrations on unstimulated and stimulated HUVEC cells in control I set it aside and plated it. Antibody-treated cells were added only to stimulated wells.

7. Add a 0.4 ml aliquot of each cell suspension to a glass tube to determine the input CPM was used to determine.

8. The assay plate was incubated for 30 minutes at 37°C.

9. Place the remainder of each cell suspension and assay plate in a water bath to cool for 20 minutes. Rejected.

10. Place the cell suspension on a cooled plate as above for the 37°C plate. I plated it with The plate was incubated at 40°C for 30 minutes. Ta.

The remaining steps of this assay were performed as described in Section A, steps 11-15 above. However, in step 12, the plate was left for 15 minutes instead of 30 minutes.

As shown in the results shown in Figure 2A, all monochromes are specific to SLX. null antibodies 5NH-3, PH6, 5NH-4 and C3LEX-1 at 37°C. When incubated in significantly blocked the binding of HL-60 cells to 1β-stimulated HUVEC. Le Monoclonal antibodies (PH2 and 5HI) specific for It is not a harmful factor. Thus, the ligand for ELAM-1 is a cell surface sugar protein. sialylated Le” antigens found in proteins or glycolipids or similar Contains a structure.

When incubated at 4°C (Figure 2B), antibodies FH6 and 5 NH-3 (both IgM) enhanced binding. In these tests, the well There appears to be significant aggregation of HL-6 (11 cells) in the cells, which supports this observation. can be explained.

In this set of experiments, LEC11 cells (which are ELA of LEC12 cells (which express SLX) and LEC12 cells (which express Le'') SLX and the unsialylated form Le” block M-1-mediated adhesion. The ability of monoclonal antibodies to be specific for.

Prepared as described. Two sets of triplicate wells served as controls. each Seven triplicate specimens on the plate were I in a volume of 0.5 ml of EGM-MV. Stimulated with L-1β at 30ug/ml. Cells were stimulated for exactly 4 hours. in general 5tanley et al., J. Biol. Chem. 263:11374 (19 88), LECII and LEC12 cells are P,5tanle Provided by Dr. Y. They are ribonucleotides and deoxyribonucleotides. Reotide (Gbco), penicillin (100 units/ml)/streptomycin (100 units/m1) (lrvineScientific), L-glutami (2mMXfrvine 5cientific) and 10% FBS ( Suspension culture in complete aluminum containing 1 ton of Haze Proliferated. The monoclonal antibodies used in these experiments were Are listed. They include: FH[3,5NH-3,5H- 1, PH-2, 3NH-4 and C5LEX-1° procedure 1. Harvest LECll and LEC12 cells, rinse in CRPM Ta. Viable cell counts were performed using Trivan blue. 3X10’! 2x cells IOml test tube, and 300μI of "CrC450uCi) (New England Nuclear) was added to each tube.

Incubate the test tube for 1 hour at 37°C with gentle agitation. Ta.

2. Wash radiolabeled cells three times in cDMEMO and place into one tube. I pooled. They are then centrifuged and incubated 4X10' in the same medium! cell/ Resuspend in 1 ml.

3, 1.6 mI culture supernatant of each monoclonal antibody, and 1.6 [11 1 purified C3LEX-1 (15 ug) was added to separate tubes.

3.2 ml of medium was added to control tubes.

4. 4. The cell suspension was added to the test tube containing the monoclonal antibody and 400 μm Added to control tube. Incubate the tube for 20 minutes at 37°C with gentle agitation. Cubated.

5. Remove the stimulated HUVEC assay plate from the incubator. The wells were then washed once with cDMEM and the medium removed from the wells using a Pasteur pipe. PET removed several wells at a time.

6. Place the cell suspension and assay plate in a water bath to cool for 20 minutes. .

7. Add 0.4 ml of cell suspension to each well of the assay plate described above. Ta. Control cells were plated on unstimulated and stimulated HUVEC. did. Antibody-treated cells were added only to stimulated wells.

8. Add a 0.4 ml aliquot of each cell suspension to a glass tube to determine the input CPM was used to determine.

9. The assay plate was incubated for 30 minutes at 37°C.

The remaining steps of this assay were performed as described in Section A, steps 11-15 above. However, in step 12, the plate was left for 15 minutes.

As the results shown in Figures 3A[g and 3B show, monoclonal antibody 5NH -3, PH6,5NH-4 and C5LEX-1 (all specific for SLX) ELAM receptors stimulate IL-1β stimulation of HUVECs. Significantly blocked binding of LECII cells. L that does not express the SLX epitope ECl2 cells did not bind to activated endothelium. Le’ (PH2 and A monoclonal antibody specific for 5HI) has a small inhibition of LECII binding. caused harm.

Further evidence that SLX is the major ligand of ELAM-Rusebuter is that Provided by removing sialic acid from LECl1 and HL-60 cells. It was. In these experiments, LEC11 and 112 cells were was processed during 5ICr labeling in Clostridium pe. Shown in Figure 4. Our results show that sialidase induces LECII and HL-6 into activated endothelial cells. has been shown to reduce adhesion of 0 cells by 70-85%.

This example is SLX, Le'' or similar but not identical compounds. Various biosynthetically produced glycosphingolipids with terminal carbohydrate units The preparation of liposomes containing the compound is described. By treatment with IL-1β HL expressing SLX to endothelial cells stimulated to express ELAM-1 SLX or pseudo-SLX to block binding of -60°C cells and LECII cells. Indicates the ability of liposomes to contain.

material The glycosphingolipids used in this experiment are shown in Table 1.

They are Biomenbrane! n5tituto, Ciato, Washington obtained from a plant and purified or produced biosynthetically and analyzed by NMR and mass spectra, Hakomori, S., 1. et al., J., Bia l, Cheffi.

259: 4672 (1984), and Fukushj Y, et al., J. Biol. Chem.

259: 10511 (1984) (these are added here by citation) ). 3-diLe’″ (SLX ) used the coio205m cell system as the enzyme source and SH as the substrate. It was synthesized enzymatically by adding a sil residue. diLe without fucosylation ” was similarly synthesized using nLc6 and cell line NCI H-69 as substrates. Ta. See, Holmes et al.

It was produced by chemically removing the terminal sialosyl residue from SH.

Liposomes containing glycosphingolipids were prepared as follows.1 00 μg of glycolipid to 300 μg in chloroform-methanol (2:1) of phosphatidylcholine (Sigma, egg yolk) and 500 μg of cholesterol (Si gma) and the whole solution was washed with N2 into a 15 ml screw tube. Evaporate to dryness in a tube with a fitted lid.

Serachi> coated 48-well assay plate (C.

HUVECs from passage 3 grown to full growth on 5 tar) were used as a source of endothelial follicles. and used it. Plates were prepared as described above.

Two sets of triplicate wells were left unstimulated. 14 3 replicate test items Stimulate with IL-1β at 30°C g/ml in a volume of 0.5 ml EGM-MV. It was intense. Cells were stimulated for exactly 4 hours. HL-60 cells and LECI+ cells Cultured as described above.

procedure 1. One 48 plate containing HUVECs grown to confluence on Seratin Remove the Co5tar cluster dish from the incubator and place each Remove the medium in the wells with a Pasteur pipette and add 0.5 ml of fresh EG. Replace with the same medium containing M-MV or 30°C g/ml IL-1β, then and returned the plate to the incubator for 4 hours.

2. HL-60 and LECII cells were obtained and cultured in CRP tL=fIO. Washed with. Viable cell counts were performed using trypan blue. 6 for each cell type XIO'' cells were radiolabeled as follows.

3 x 101 cells of each type were placed into 2XIOml test tubes and 300μm ”Cr (450μCiXNew England Nuclear) was used for each test. Added to test tube. Ink the test tube for 1 hour at 37°C with gentle agitation. was developed.

3 Radiolabeled HL-60 and LECl1 cells were incubated three times in CRPMI. Washed and pooled into one tube. Then centrifuge them and and resuspended at 2XIO' cells/ml in the same medium.

4. Remove the heated HUVEC assay plate from the incubator and set it aside. Then fill the wells with RPM1 containing 5 mg/ml bovine serum albumin (BSA). Washed twice with 1640.

5. Liposomes were prepared as follows: 100 ml of the evaporated precipitate was Dissolved in water ethanol and sonicated for 2 minutes.

2 ml of PBS was slowly added to the test tube over 2 minutes while being sonicated. child The stock was diluted 1:10 in RPM 11640 immediately before use and 50 The stock solution of μm BSA is 100mg/m! 1 ml of each diluted liposo A final concentration of 5 mg/11 BSA was added to the sample.

6. Pour the medium from the wells of the assay plate several times at a time using a Pasteur pipette. and 0.3 ml of the liposome suspension was stimulated with 6 IL-1β. into each well of the assay. In control wells, wells containing liposomes were added. containing ethanol, RPMII640 and BSA at the same concentration as in the Added liposome buffer. Control buffer contains unstimulated HUVEC and stimulated HUVEC. It was plated on a HUVEC. A sample containing liposomes was stimulated with a was added only to the wells.

7. Incubate the plate at 37°C for 40 minutes, then incubate the plate with 50IIll Add H(:r-labeled HL-60 or LEC11 cells to the wells of the assay. did. Each cell line was assayed in triplicate for each liposome preparation.

Each cell! ! ! Three 50°C aliquots of the suspension are used to determine the input CPM. into glass tubes and incubate assay plates for 30 minutes at 37°C. was developed.

8. Unbound cells are passed from the wells of the assay plate by systematic resuspension. Using a tool pipette, then remove by adding and removing 0.7 ml of medium. I left. Remove all medium from the wells and add 0.125M T r s, 2% SDS and 10% glycerin were added (0.3 ml). play The cells were left for 15 minutes and then 0.5 ml of dH,O was added to each well.

9. Resuspend the fluid in each well and transfer to a glass tube. Pipette Insert the tip of the tip into the tube. Tube containing the input CPM sample was counted with a gamma counter.

Divide the bound CPM in each well by the input CPM for each sample to calculate the combined % was determined. Plot the mean and standard deviation of assay points for triplicate tests did.

As shown in Figure 5, the terminal end containing SLX (S-d Liposomes containing selected glycolipids with columns are active at 4°C. The adhesion of HL-60 cells to transformed endothelial cells was dramatically inhibited. Le’ (di -Le') or other related structures (Table 1) Somes showed minimal inhibition that was independent of the structure of the carbohydrate groups. A similar result is L Obtained by attachment of ECII cells. When the experiment was carried out at 37 °C, HL- 60 cells attached to glycolipids with SLX structure (S-d iLe”, 70%). and to a lesser extent also by liposomes containing Le” (di-Le”, 40%) was reduced by liposomes containing (di-Le”, 40%); It has been suggested that it can interact with ELAM-1 with lower affinity. Ru. These experiments demonstrate that biosynthetically produced SLX or similar pseudo-S When incorporated into liposomal compositions, LX compounds can be used, for example, to It can act as a therapeutic compound for the reduction of blood cell infiltration.

Jurkat cells are activated via ELAM-Rusebuter. In contrast to the attachment of HL-60 and LECz to endothelial cells, V- IL- through CAM (endothelial cell)-VLA-4 (Jurkat cell) adherent pair 1 binds to activated endothelial cells. Adhesion of Jurkat cells to SLX-containing α-subunit of integrin molecule VLA-4 was completely inhibited by a monoclonal antibody against As this result proves Furthermore, inhibition of SLX liposomes in HL-60 and LECII cells inhibited endothelial cells. Rather than the steric effects attributed to liposome binding, SLX liposomes -1 results in inhibiting attachment by directly competing with the binding site of the ligand. The theory is supported that antibodies against SLX are mediated by GMP-140 on activated human plates. impede cooperation In this example, GMP-1 of HL-60 cells to activated human platelets. 40-mediated attachment, specifically for SLX and unsialylated Le” Determine the potency of different monoclonal antibodies in HL-60 cells as described above, and was used as a cell source with Jurkat cells as control without ligand and used it. Monoclonal antibodies 5H-1, FH-2, 5NH-4 and C3 LEX-1 was also mentioned above.

2.0. g; Sodium citrate 2.49g; and citric acid 1.25g; d 6 parts blood/1 part anticoagulant into a syringe containing 100 + nl at 820 It was extracted based on the solid agent ratio.

Platelets were separated by differential centrifugation as follows: blood was centrifuged at 800 rpm ( Centrifuge at room temperature (90xg) for 5 minutes. Collect the supernatant, and 120 Centrifugation was performed for 6 minutes at Orpm (4 ooxg). Remove the supernatant liquid, and Centrifugation was performed at 000 rpm (1200×g) for 10 minutes. tie platelet button Load - HEPES buffer, pH 6,5 (Na C18, Og: to, C1, 0.2g; N a Hz PO2・H200,057g; Mg C12 ・6 H200, 184g; NaHCOs O, jg: Dekitrose 1 ．． Og: and HEPES 2.383g + DI water to make 1 liter, (adjust to pH 6.5 with NaOH) twice, then once in PBS. Washed. Platelets were suspended in PBS at a concentration of 10'/ml.

2. Approximately 20 minutes before the final resuspension of platelets, the 48-well plate was % gelatin and incubate for 15 minutes at 37°C. I turned on. Excess gelatin was removed with a pipette just before addition of platelets. platelets Platelet suspension at 0.25 units of thrombin/ml (SigmaT-6759) was activated by the addition of Platelets were left at room temperature for 20 minutes.

To prepare 36-bound and activated platelets, a 300 μm platelet suspension was added to each well of the gel-coated plate. Place the plate at 37°C. Incubate for 15 minutes at 800 rpm (90 x g). Rotated for 2 minutes. Remove unbound blood by washing the plate three times with PBS. The platelet was removed.

4. Platelets have highly reactive Fc receptors, so they do not aggregate from antibody m* In order to prevent the absorption of IgG, platelet Fc receptors are Blocked: ff1H mouse IgG6/32 (IgG2.) 27 Agglomeration was achieved by heating to 63° C. for 5 minutes at mg/ml. 300m Platelets were coated at 20 gg/ml in a heated preparation of 1 in PBS. were added to each well of the plate. Ink the plate for 15 minutes at 37°C. and then washed with PBS.

5. Harvest 8L-60 and Jurkat cells and wash in CRPMl did. Viable cell counts were performed using trypan blue, and winter-type 3X10” Place the cells in 2X10ml test tubes and 300μm S1Cr (45 0 μCi) (New England Nuclear) was added to each test tube. . Incubate the tube at 37°C for 1 hour with gentle agitation. did.

6. Wash the radiation-injured cells three times in CRPMI and place in one test tube. I pooled inside. Then, centrifuge them and place 2 x 10'' cells into the same medium. cells/m extracellularly suspended.

7. 1.6 ml of culture suspension of each monoclonal antibody, and 1.6 ml of sperm The prepared C3LEX-1 (15 gg) was added to separate test tubes.

1.6 ml of medium was added to control tubes.

8. 100 μm of Jurkatm cell suspension (containing 4X to’ cells) Labeled HL-60 was added to each tube containing the monoclonal antibody. . Incubate them at 37°C for 20 minutes with gentle agitation. I turned on. After this incubation period, 0,3 ml of each cell suspension (7,5 X10' cells), as described above, containing bound activated platelets. were added to each well of the assay plate.

9. Centrifuge the assay plate at 90% g for 2 minutes, then incubate for 5 minutes at room temperature. I did an incubation con. Invert the plate into a radioactive waste container and Remove unbound cells by blotting the plate onto a towel. was removed from each well of the SayPlate. Carefully add 300 μM PBS to each well. Add the solution to the wafer by inverting the plate and blotting. The tube was washed three times. All of the medium was removed from the wells and 0.125 molar Added 0.3 ml solution of Tris, 2% SDS and 10% glycerin . Leave the plate for 15 minutes, then add 0.6 ml dH20 to each well. Ta.

10. Resuspend the fluid in each well with a pipette and transfer to a glass test tube. did. Place the tip into the tube. Gun the test tube containing the one containing the input sample. I counted it with a macounter. The bound CPM in each well was measured for each sample. and divided by the input CPM to determine the % combined. Enter the input CPM in step 8 It was determined by counting the final coat of 0.3 ml of each cell suspension.

As shown in the results shown in Figure 6, monoclonal antibody 5 specific for SLX NH-4 and C3LEX-1 influence GMP-140 on activated platelets. Binding of HL-60 cells was blocked.

Monoclonal antibodies specific for "Le" (FH2 and 5H-1 are also available for this purpose) binding was blocked, but to a lesser extent. As this example suggests, Both SLX and Le'' can be ligands for GMP-140. However, the SLX structure has a higher affinity for GMP-140 than the Le” structure. This example shows that GMP-14 can be isolated by treatment with thrombin. F (L-60 cells expressing SLX) to platelets stimulated to express 0 and liposomes containing SLX or pseudo-SLX that block PMN binding. prove the ability of the team. This assay is generally described by Larsen et al., Ce1l 6 3: 467-474 (1990) (add this here by citation) The protocol described in was followed.

material Glycosphingolipids were prepared as described in Example II. small blood Plates were prepared as described in Example ■, except that Fc receptor blockade was No packing was performed. HL-60 cells were prepared as described above.

PMNs received heparinized vacutainers from volunteer donors. tafner) from 50 ml of whole blood drawn into a tube, the tube being inverted. Mixed blood. All steps were performed at 22-24°C. 2511 each blood in 15 ml of Mono-Poly Resolvi ng Medium) (Flow-Labs). 800x tube g for 25 minutes, then centrifuged at 1300×g for an additional 25 minutes. Excluding PMN layer and placed into a clean 50cc centrifuge tube. 20mM HEPES 30m containing (Gtbco) and 0.2% glucose (Fisher) 1 bank of balanced salt solutions (Gi bc o) are added to the vessels, and then these was centrifuged at 1900×g for 3 minutes. PMNs were incubated at 1900×g in the same buffer for 3 Washed three times by centrifugation for minutes.

PMNs were counted with a hemocytometer, resuspended at 2 x lO'/ml, and used. It was kept at room temperature until

procedure 1, 20 μl of activated platelet preparation into 28 1.5 ml bottles. into each of the Luff tubes (samples of 14 duplicate experiments).

2.20 μm diluted liposomes at 1 Gag + 5 μg or 2 μg, Alternatively, 20 μl of control buffer was added to the appropriate tube of each duplicate experiment.

3□ Platelets were incubated with liposome preparation for 20 minutes at room temperature. .

4. One set of neutrophils or HL-60 cells each at 2 x 10' cells/ml. It was added to posome-treated platelets. 20 μl of cell suspension was added to the tube.

5. The tube was mixed and left at room temperature for 20 minutes. Then put them on a hemocytometer and cells are positive (2 or more attached platelets/cell) or negative (2 or more attached platelets/cell). The platelets were scored as less than 2 platelets/cell attached.

Selected glycolipids with terminal sequences containing SLX as shown in FIG. Liposomes containing S-diLE” (Table 1) can be transferred to activated platelets. dramatically inhibited the adhesion of HL-60 cells. Le” (d 1-Le”) also Glycolipids (Table 1) with other related carbohydrate structures structure-independent), showed minimal inhibition. Similar results using PMN cell attachment (Figure 8). As these experiments show, biosynthetically produced S When incorporated into liposomal compositions, LX or similar pseudo-SLX compounds can be used, e.g. , a therapeutic compound for reducing leukocyte binding to platelets at inflammatory sites. can work as.

Example■ Hexasaccharide SLX promotes the binding of neutrophils to platelets. -1-1-1-1-1'--1-1-1-1-10 In the example of Also compare the abilities of LX with those of Penta and Hexasa Tucarid. Briefly Afterwards, platelets and neutrophils were isolated as previously described. Activate platelets with thrombin Neutrophils were incubated with dilutions of various oligosaccharides in (X). Effect of saccharide on neutrophil adhesion to added and activated platelets It was determined. The oligosaccharides used were: SLX (hexa), NeuA, cα2.3Ga Iβ1,4 (α1.3Fuc)GlcNAcβ1 ．． 3Galβ1.4G1cm0-CHz CH2SiMex (Gifu University Hase provided by Professor Gawa), SLX (penta)NeuAcα2,3Galβ1,4 (Fucαl, 3) GIcNacβ1.3Galβ, and SLX (tetr a), NeuAcα2゜3Ga Iβ1.4 (αt, 3Fuc) G1cNAc a procedure 1. Platelets were separated as described above and treated with thrombin at 0.250/ml. Activated by incubation for 20 min at room temperature at final concentration. (2×10’/ml).

2. Neutrophils were grown in mono-poly lysis medium (Ficoll-Hypa) as described above. Heparinized blood was placed on top of que-Flow Laboratories). layer, then centrifuge for 25 minutes at 2000 rpm, then 2500 rpm. The cells were separated by centrifugation for an additional 25 minutes at m.

3. For assay, 201. tl platelet suspension (2X 10＠;’ ml ) was placed in an Eppendorf centrifuge tube. Equal volume of oligosaccharide preparation is 50 At concentrations of 0 μg/+nl to 2.0 μg/ml or glycolipid Posome preparations (prepared as described above) were added at 2 μg/ml-0.25 μg/ml. 1 concentration and the tubes were left at room temperature for 20 minutes. then 2 Add a 0 μm neutrophil preparation (2X 10”/ml) and bring the tube to room temperature. The mixture was left for an additional 20 minutes.

Adhesion of activated platelets to 44 neutrophils was assayed microscopically. 100 Neutrophils were evaluated. have two or more platelets attached to them If less than 2 platelets are attached, then negative I attached the core. Calculate the percentage of cells with 2 or more bound platelets. Ta.

The average results of three identical experiments are shown in Table 2.

Table 2 Amount required for 50% inhibition Oligosaccharide (μmol) SLX (hexa) 1.8 SLX (penta) 2.2 SLX (t e t r a) 54.0Le’ 43.0 GMP-14 of neutrophils to thrombin-activated platelets as shown in Table 2. nearly 20 times more than 5LX-hxa saccharide to inhibit 0-mediated binding by 5%. The above 5LX-tetrasucharide is required. Tetra Satsuka required The amount of Lido is equal to that required for a similar degree of inhibition when using non-sialylated Le. Almost equal. Pentasacucalide is 50% inhibited by hexasacucalide. gave 50% inhibition at a concentration similar to that required for This indicates that the minimum structure of the molecule is close to that of a pentasaccharide.

This example describes experiments testing various glycolipid structures on liposomes. do. In particular, SY2, i.e. fucose is the ultimate Gl as in SLX. cNA, attached to the penultimate GlcNAc instead of attached to c. The sialylated polysaccharides attached were tested. Platelets and neutrophils were collected by the method described above. separated. Platelets were activated with thrombin and then treated with liposomes prepared as described above. Incubation with dilutions of various glycolipids embedded within the Ta. Glycolysis to add neutrophils and adhesion of neutrophils to activated platelets The effect of Pido was determined.

The structures of the various glycolipids tested are as follows: 5DiY2°NeuG ca2.3Galβ1.4 (Fucα1.3) GlcNacβ1.3Galβ1 ．． 4 (Fucαl.

3) G1cNAcβ1.3Galβ1.4Glcβ1. ICer; SLX, NeuGca2.3Galβ1゜4(Fucα1.3)G1cNAcβl, 3Galβ1.4G1cβ1. fcer; SY2. NeuGca2゜3 Galβ1.4G1cNAcJ1.3Galβ1.4(Fucα1.3)G1 cNAcβ1.3Galβ1゜4Glcβ1. ICer; SH, NeuG ca2.3Galβ1.4GlcNAcβ1.3Galβl.

4G1cNAcβ1.3Galβ1.4G1cβ1. ICer; SPG, NeuGca2.3GalβI.

4GlcNAcβ1.3Galβ1.4GICβ1. ICer.

The results of two identical experiments are shown in Table 3.

Table 3 Amount required for 50% inhibition Oligosaccharide (μmol) SY2 (Experiment 1) 0.325 SY2 (Experiment 2) 0.345 SLX (hexa) 0.30 SdiY2 0.36 Does not inhibit SPG Does not inhibit SH As these results show, SY2 is as good as SLX and 5DiY2; Inhibited GMP-140-mediated adhesion of neutrophils to thrombin-activated platelets .

This example demonstrates the affinity of sialylated Le'' (SLX) for GMP-140. However, the terminal sialic acid is N-acetyl neulaminate (NeuAc) or N-group. Recall Neulaminae) (NeuGc) is the same regardless of whether it is prove that All materials were prepared as previously described. Platelets and neutrophils Spheres were separated by the method described. Platelets are activated with thrombin and then lipolytic Incubated with various dilutions of glycolipids contained within the . Addition of neutrophils and glycolysis of neutrophils to activated platelets The action of lipids was determined.

Synthetic SLX (Neu, A, c) and Soaryl purified from bovine red blood cells Preparation of SLX prepared by enzymatic fucosylation of balagloboside SLX ( Table 4 shows the results of experiments that directly compared NeuGc).

Table 4 Amount required for 50% inhibition Oligosaccharide source (μmol) SLX (bovine red blood cells) 0.74 (NeuGc) SLX (synthesis) 0.67 (NeuAc) As these results show, 5LX-hexasaccharide has sialic acid that is neutralized by NeuA. Whether c or NeuGc, there is a preference for thrombin-activated platelets. GMP-140-mediated adhesion of mesospheres was equally well inhibited. As this result shows, N-acetyl or N-glycolyl derivatives of sialic acid are recognized by GMP-140. be recognized.

Similar results were obtained using ELAM-1.

Various glycolipids were also tested in the same assay. Glycoli tested The structure of Pido is as follows: 5LX (hexa), NeuGcα2゜3G a　lβ1.4(Fucα1.3)GlcNacβ1.3Galβ1.44Gl cβ1. ICeramide;a2. 3 SLX car, NeuAca2 ．． 3Galβ1. 4(Fucα1.3)GlcNAcβ1゜3Ga　β 1.4Glcβ1. ICeramide: a2.6 SLX cer, N euAcα2.6Galβ1.4 (fucα1.3)G1cNacβ1.3Ga lβ1.4G1cβ1. ICeramide: SH.

NeuGcα2.3Galβ1. , 4GlcNAcβ1.3Galβ1.4Gl cNAcβ1. ,3Galβl.

4G1cβ1. ICeramide.

This example shows that synthetic SLX binds ELAM-1 and directs it to activated endothelium. demonstrated to inhibit neutrophil adhesion. This example also shows a chain of sialic acids. affects binding to ELAM-1.

Two synthetic compounds were prepared. One, as in naturally occurring SLX, It contained α2,3-chain sialic acid. The second is the nature of the linkage to receptor binding. In order to examine the importance of quality, it consisted of α2°6-chain sialic acids.

Liposomes were prepared by adding 12 μm anhydrous ETOH to a container and placing it in a 50°C water bath. Prepared by brief warming and sonication for 2 minutes. 238μm Slowly add warmed phosphate buffered saline (PBS) to the vessel while sonicating. and sonicated for an additional 10 minutes. The final concentration of stock liposomes is It was 400 μg glycolipid/ml in 5% ETOH/PBS.

procedure 1, HUVEC, PMN, and liposomes were prepared as described above.

2. Remove the stimulated HUVEC assay plate from the incubator and The sample was then transferred to RPM11 containing 5 mg/ml bovine serum albumin (BSA). 640 twice.

3. Dilute the liposome stock in HBSS/BSA buffer and then make an equal solution. were prepared: 40μg/11.30ug/mL, 15ug/ml, 7.5μg/ ml, 3.75 μg/1Ill and 1.87 μg/ml. Similar dilutions in PB A control stock consisting of S-5% ETOH was prepared.

4. Pour the medium from the wells of the assay plate several wells at a time using a Pasteur pipette. It was removed using the file.

5. Duplicate on the assay plate stimulated 0.05+ nl of each liposome suspension. were added to wells in replicate experiments. in control wells and in wells containing liposomes. Liposomes containing ethanol, HBSS and BSA at the same concentration as in Buffer was added. Control buffer unstimulated HUVEC and stimulated HUVE Played on C. Only in wells stimulated with samples containing liposomes Added.

6. Incubate plates at 37°C for 40 minutes, then add 50μ m PMNs were added to the wells of the assay. The final concentration of cells was 5 in 100 μm. It was a X10S well.

7. Return the assay plate to the incubator (5% CO2, 37°C) for 8 minutes. did.

8. Remove unbound cells from the assay plate by systematic resuspension of p200 Using a channel pipette, then add and remove 0.2 ml of medium. It was removed by

9. Remove all of the medium from the wells and add 50 μl of solubilization buffer. Ta. This is a citrate buffer containing 0.1% No-40 detergent (24,3 ml of 0.1M citric acid, 10.5g/500ml: 25.7ml of 0.1M citric acid, 10.5g/500ml: 2M sodium phosphate, 1.4.2/500ml and SQH, O at IQOm It consisted of l).

10. Incubate the plate on a rotary shaker for 10 minutes, then 0.05[[+1 of 0PDA solution (8 mg of 0-phenylene-diamine, Si gma Catalog No. P-1526, 8ml of 30% H2C.

and 10 ml of citrate buffer (as above)] were added to each well. child The reaction was allowed to proceed for 15 minutes, then 25 μM of 4N sulfuric acid was added to each well to incubate the reaction. response was stopped.

118 reagent in a volume of 100 μm of solubilization buffer and 0 PDA solution in a volume of 50 μm. 4 by mixing with sulfuric acid.

12. Remove 100 μl of supernatant from each of the 2 wells and place in a flexible E Transferred to LISA assay plate (Falcon). Spectrophotometrically measure the plate was scanned within 30 minutes at 492 nm.

The results of the two experiments are presented in Table 5 below.

Table 5 (2,6)SLex (2,3)SLex concentration average average 1 20ug/ml O, 6630, 156215ug/ωl O, 6360, 27037, 5μg/mouth 1 0.602 0.3594 3.75μg/ml O, 6550, 48351, 87a g/ml O, 6900, 5806, 4 7ttg/ml O, 6950, 64270 μg/ml o, 710 0.7 16 control + IL-], 8 control-IL-IB concentration average average 1 20u g/ml O, 6570, 010215ug/ml O, 7400 ,01037,5μg/ml O,6580,01343,A5u g/ml. 0.698 0.0095 1.87u g/ml O,7250,0146 ,47μg/l1ll O,7820,01870μg/+ml 01708 As these results show, the synthetic α(2,3) sialyl Le''' However, liposomes that do not contain α(2,6)sialyl Le" are ELAM-1 Inhibits neutrophil attachment to activated endothelium in a dependent assay. child Thus, the α2,3 linkage of sialic acid is required for recognition by ELAM-1. I think that the. Moreover, as the results show, synthetically produced oligosaccharides, α(2 , 3) Sialyl Le” binds to ELAM-1 and promotes activation of endothelium. Block the union of the middle sphere. Similar results were obtained for GMP-140.

It is shown in FIG. Therefore, this compound or derivatives of this compound have the potential to constitutes a candidate for anti-inflammatory drugs.

A practical example of this is the β-galactose inside sialylated Le1 of HL-80JI cells. The main chain sugar linkage is sensitive to endo-β-galactosidase cleavage. We describe experiments to determine whether endo-β-galactosidase is It cleaves the internal β-galactose chain within the lactosaminyl structure, but Ga1N When AC is bound to mannose (core structure), it does not cleave β-gal. This is an enzyme known as

procedure Platelets were isolated by the method described above and activated with thrombin. rL-1β The activated HUVECs were prepared as described above. Cultured HL60 cells Blood treated with endo-β-galactosidase and activated as described below. Determining the effect of enzyme treatment on GMP-140-mediated attachment of HL6 ([1) cells to platelets. Ta.

Enzyme treatment of HL60 cells was performed as follows: 12.4×10” cells were Hang balanced salts containing 0mM HEPES and 0.2% glucose Washed twice with solution and then once with normal physiological saline. End-β- Galactosidase (0,1 units, rcN Chemicals.

[NC, Irvine, California] in 200 μl of normal saline solution. and dissolved in 200 μl of sodium acetate buffer (pH 6,01). 20 Add 0 μI (containing 0.05 units of enzyme) to HL60 cells at 3XlO' and a similar number of cells using 200 μm acetate buffer as a buffer control. added to the cells. Both tubes were incubated at 37°C for 60 minutes with gentle agitation. Cubated. The tubes were then chilled on ice and the cells were incubated with HEPES and washed 3 times in HBSS containing glucose, then counted and 2 It was suspended at x 10'/ml.

For the GMP-140 assay, add 20 ml of Tyrode-HEPES buffer ( pH 7.2) was placed in an Etzbendorf tube. Same volume of activated platelets (2xlO”/ml) Add Oyobi HL60m cells (2xlO@/ml) After mixing, the tubes were left at room temperature for 20 minutes.

Regarding the adhesion of platelets to HL60 cells and the adhesion of activated platelets to neutrophils Microscopic evaluation was performed as described above.

As these experiments show, treatment of HL80 cells with endo-β-galactosidase (1) The ability of HL60 cells to bind to thrombin-activated platelets. was inhibited by 87.5%, and (2) HUV activated by IL-1β. EC was inhibited by 70% at 4°C. Thus, for GMP-140 The tetrasaccharide ligand containing the smallest SLX is probably more sensitive than mannose. Rather, it is bound to lactose or polylactosaminyl structures.

Example X■ In this example, fucosylated polysaccharides block neutrophil binding to platelets. The ability of fucosylated polysaccharides to inhibit GMP-140-mediated adhesion was compared with that of non-fucosylated polysaccharides. The comparison was made with that of the hexasatsucharides SLX and Le. Platelets and neutrophils were isolated by the method described above. Activate platelets with thrombin , followed by incubation with various oligosaccharide dilutions. Add neutrophils, The effect of saccharide on neutrophil adhesion to activated platelets was then determined.

The oligosaccharides used were: natural polysaccharides and their fucosylated derivatives ( The preparation of both is described below).

SLX hexasatsucharide, LNF II [(Le”) and LNF 1 (structure (mentioned above).

The polysaccharide containing the linear core structure of SLX is enzymatically transformed into a polysaccharide containing polyvalent SLX. Converted by fucosylation. Natural polysaccharide type Ia is described by Jennings et al., Bioc hem, 221258-1263 (1983) (which is incorporated herein by reference). Streptococci of group B as described in ccus). A suitable bacterial strain is American type culture. Collection (AmericanType C1ture Co11e) ction) and Trustee No. 12400.31574゜124 01, and 31575.

To prepare the fucosylated polysaccharide, 1 mg of natural type Ia polysaccharide was mixed with 1 mol of 6 μm Manganese chloride, guanosine 5'-diphosphate β-L-fucose and radioactive training cer (specific activity 1.82X lO'cpm/μmol), 0.9 in 90μm water μmol, and 137 μl of water were dissolved in a mixture. To this, Pr1eels et al., J. Biol, Chem, 2561045&-10463 (1981) (this isolated from human milk, as previously described in A solution of 100 μl of separated 3/4 fucose transferase was added.

The reaction mixture was wired several times with water to a membrane (100K cutoff), The retentate was then lyophilized to powder. Dissolve this solid in water and Remaining proteins were removed through an ion exchange column. fucosylated poly Radioactive fractions containing saccharides were collected and lyophilized. by radioactive worker As measured, approximately 50 available side chains were fucosylated.

procedure Platelets were separated as described above and treated with thrombin at room temperature at 0.25U/ Activated by incubation for 20 min at a final concentration of ml ( 2×10”/ml).

Neutrophils were grown in mono-poly lysis medium (Ficol 1-Hypaq) as described above. ue.

Layer the heparinized blood on Flow Laboratories. , then centrifuged at 200 rpm for 25 minutes, then 2500 rpm for an additional 25 minutes. Separated by centrifugation at pm.

For the assay, a 20 μm platelet suspension (2X 10’/ml) was I put it in a ndorff tube. Equal volumes of oligosaccharide preparations from 500 μg/ml to were added at a concentration of 2,0 μg/+nl and the tubes were left at room temperature for 20 min. . Then add 20 μl of neutrophil preparation (2X10'/ml) and close the tube. It was left at room temperature for an additional 20 minutes.

Adhesion of activated platelets to neutrophils was assessed microscopically.

100 neutrophils were evaluated. They are associated with two or more platelets If less than 2 platelets were bound, score as negative. Wearing. The percentage of cells with two or more bound platelets was calculated.

As shown in Table 6, fucosylated polysaccharides act as neutrophils for thrombin-activated platelets. GMP-240-mediated binding of the globules was inhibited very efficiently.

50% inhibition was achieved with amounts less than lμg/l111. This is a natural polysaccharide comparable to the 20 μg/layer I required for SLX and for a similar degree of inhibition. Comparable to 8 μg/ml of hexasaccharide.

Table 6 Amount required for 50% inhibition Oligosaccharide (μmol) Natural polysaccharide 15 Fucosylated polysaccharide 0.7 SLX Hexasatsucarid 8 LNF II (Leri 35 LNF I No inhibition An example of this is mAb in an animal model of polysaccharide-induced death. P2H4 (described in pending U.S. Patent Application No. 07/645.878) Figure 2 shows the effect of murine Ig03K, a functional anti-human ELAM-1 mAb. . P2H4 is known to cross-react with the rat equivalent to ELAM-1. Therefore, a rat strain was selected.

Materials and methods LPS from Escherichia coli 0112:84 (Sigma) was added to a single aliquot one day before use. Dissolve in sterile pyrodiene-free salt solution at a concentration of 5 mg/ml from the lot. Prepared by: The solution was crushed for 30 seconds using a Tekmark Sonic crusher. Sonicated on ice. Just before use, sonicate the material a second time for 30 seconds. Ta. Female Lewis rats weighing 200 g ± 10 g were placed in a Charles Rive Breeding Labs and kept for at least 7 days after purchase. I grew it (to grow to 1m). Unless otherwise specified, multiple groups (10 animals) were used. used. All reagents were injected parenterally through the tail vein. As a negative control, sterile LPS-free 1r saline solution or murine Ig03 with myeloma protein ( J 606, low pyrodiene - <2 ng/mg protein).

result The P6E2 dose/schedule protocol is based on P6E2 administered prophylactically to rats. It was obtained experimentally from pharmacokinetic data obtained by the inventors with 2H4. "Minimum j L D. . The amount was experimentally determined to be 7.5 mg/kg for these rats. It was.

In one experiment, 1 hour before challenge. Treated with Omg/kg Ta. Boost was administered 3 hours after challenge. 4/10 treated animals Survived against LPS. On the other hand, all 10 animals (injected with saline solution) vs. Teru died. During the 24-hour observation period, animals that survived were treated with LPS. The animals showed almost no clinical symptoms characteristic of these animals.

We then determined that the lomg/kg dose used in the first experiment Attempts to administer (1) two times higher doses and (2) one order of magnitude lower doses of PSE2. Ta. These results show that P2H4 has a significant effect, with 80% % of animals survived at +omg/kg dose (Iglo). In addition, one animal was salt-soluble. No animals survived in the control group, indicating that the animals in this group were not "humanized". did.

Other studies were conducted to demonstrate the therapeutic value of P2H4. I, PS challenge 1 Porus administration of 10 mg/kg P2H4 before or 2.4 or 6 hours later did.

Again, 1 out of 10 animals in the group treated with saline solution and +om In the group treated with g/kg J606 myeloma protein (T=60 min) It remained behind (Figure 11). When P2H4 is administered 2 or 4 hours after LPS However, it had a protective effect.

Current opinion The protection seen with Cytel mAb P2H4 was demonstrated in an animal model of the fatal disease. This shows the importance of ELAM-1 in this study.

An example of this is sialylated Le, isolated from a biological source and introduced into ribosomes. is　x　(SLX) and sialoparagloposide (SPG) are activated [Rolling J (r.

11ing).

"Rolling" is a periodic cell-cell interaction between leukocytes and the endothelial cell wall. white blood The spheres "roll" over the endothelial cells. The white blood cells then (1) return to the circulation; , or (2) attaches to the endothelial cells and initiates the initial events that culminate in inflammation. Selek Chin describes “rolling” glycosphingolipid-containing ribosomes as follows: It was formed by 50 μg of glycolipid was dissolved in chloroform/methanol (2° 1) 150 μg of phosphatidylcholine (S i gma, egg yolk), 250 μg g of cholesterol (Si gma) and 1 mM carboxyfluorescein ( Si gma) and the entire solution in a glass tube with a screw cap. Evaporated to dryness. Add 12.5 μl of pure ethanol to each tube and briefly in a water bath. Ribosomes were prepared by warming for 2 minutes and sonicating for 2 minutes. Sonic treatment Gradually add warm PBS to each tube while controlling and sonicating for a total of 10 minutes. continued.

14. Make ribosomes I[l11 with PBS; At OOOrpm 2 Excess EtOH and carboxyfluorescein were removed by centrifugation for a minute.

Discard the supernatant and resuspend the ribosomes in 1 ml PBS and place in a hemacytometer. were counted and adjusted to 5X fO' ribosomes/mi. by IL-1 Injected into activated rabbits 4 hours prior.

lori using an intravital microscope as described by Acfrian et al. observed. The TL-1 activated rabbit mesentery was excised and I It was spread on a glass window heated to 37°C on the I microscopic stage. the mesentery , in a salt solution at 36,5 °C equilibrated with 5% COz in N2. Ribozo Mu-endothelial interactions were observed by intravital microscopy with a 50X saline immersion objective. I guessed it. Ribosomes were visualized by exposure to fluorescence. Selected intestinal veins Segments of tubules (20-40 μm in diameter) were counted over 1 minute.

result Rohit neutrophils on rabbit cells activated by IL-1 4 (Maximum amount of rolling) S L e x Ribosome 2 SPG ribosome 0 (no rolling) conclusion In addition to LAM-1 (van Adrian et al.), ELAM-1 and its Ligands can be used to control leukocyte marginatility (marginat 1on) or "rolling" It seems to be involved in existence.

Although the present invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, , it is obvious that certain changes and modifications do not depart from the scope of the appended claims. It can be done.

b Inhibition of platelet adhesion by monoclonal antibodies FIG, 6° Liposome concentration (uq/ml) Liposome concentration (4q/ml) FIG. Survivors (%) hC〃 abstract The present invention is directed to reducing or suppressing inflammation, as well as inflammation mediated by cell-cell adhesion. The present invention relates to compositions and methods for treating symptomatic disease processes and other pathological conditions. Se selectively binds to a cretin receptor and has at least one selectin binding component. Provided by the present invention are compositions having the following properties: The selectin binding component has the general formula: RI-Ga 1βl, 4 (Fucαl, 3)GlcNAc-(Rz), Here, R1 is oligosaccharide or R'! -R4C(Co□H)-, where R3 and R4 are the same or different, and -H1-CI-C8alk Kyl, -hydroxyCl-C8 alkyl, -arylC1-C8 alkyl, or -AlkoxyCl-C8 alkyl, where R2 is β1,3Gal, ai 2Man or αl, 6GalNac, and a is 0 or 1.

Submission of translation of written amendment (Article 184-7, Paragraph 1 of the Patent Act) 1 Display of patent issue PCT/US91104284 2 Name of the invention Cell-cell adhesion mediator.

3 Patent applicant Address: California, USA 92121. San Diego, Gijin Hopkins Court 3525 people Name Cycil Corporation The scope of the claims 1. selectively binds to a pharmaceutically acceptable carrier and the securen receptor comprising a compound comprising a component, said component having the formula: %formula%) R1 is a group consisting of sialic acid, an oligo ring comprising sialic acid, and a group having the formula R3 and R4 are individually the same or are different, and -H, C, -C, alkyl, hydroxy (C, -C, alkyl ), aryl-(c, -c, alkyl) and (C, -C, alkoxy) (CI Cs alkyl), or Rff and R4 together are 4- to 8-membered forming a ring, and R2 is β1,3Gat, αl, 2Man or crl, 6G alNac, and a is O or 1, and X is -H2 lower alkyl (C, -Cs), arylalkyl, hydroxy cc, -cm>alkyl or (CI　Cm)alkoxy-(c.

-C3) is alkyl) A pharmaceutical composition comprising:

2. The composition of claim 1, wherein the compound is a biological molecule.

3. The biological molecule is an oligocycle, an oligopeptide, a protein, or a lipid. , the composition of claim 1.

4. The composition according to claim 1, wherein R3 and R4 are 4- to 8-membered rings.

5. The composition according to claim 4, wherein the 4- to 8-membered ring is a monosaccharide.

6. Claim in which the sialic acid is NeuA, cα2,3 or NeuGcα2,3 1. The composition according to 1.

7. The oligo ring is NeuAc+r2,3Galβ1,4GlcNAcβ1,3 or NeuGcα2.3Galβ1,4G1cNACβ1,3, the claim Composition '#y as described in 1.

8. The composition of claim 1, wherein the compound is a polysaccharide.

9. The oligo ring is of the formula.

↑ 9. The composition according to claim 8, comprising a repeating unit having:

10, the oligo ring has the formula: 9. The composition according to claim 8, comprising a repeating unit having:

11. The polysaccharide is a group B Streptococcus fucosylated Ta-type polysaccharide; The composition according to claim 8.

12. Claim 8, wherein the polysaccharide is a group B Streptococcus type polysaccharide. Compositions as described.

13. Claim 8, wherein the polysaccharide has a molecular weight of about 5,000 to 300,000. The composition described in .

14. Claim 14, wherein said polyws comprises from about 5 to about 200 fucosylated repeat units. 8. The composition according to 8.

15, wherein said poly[r] comprises from about 25 to about 100 fucosylated repeat units; 15. The composition according to claim 14.

16. The composition of claim 1, wherein the biomolecule is a sphingolipid.

17. The composition according to claim 16, wherein the biomolecule is a ganglioside.

18. said selectin receptor is expressed on vascular endothelial cells or platelets; A composition according to claim I.

19. The selectin receptor is ELAM-1 or GMP-140. A composition according to claim 18.

20, a pharmaceutically acceptable carrier, and selectively binding to a selectin receptor A pharmaceutical composition comprising a liposome having a compound.

21. The liposome according to claim 20, wherein the liposome encapsulates an anti-inflammatory chemotherapy agent. Composition of.

22. The anti-inflammatory agent is cyclosporin A, indomethacin, naproxen, FK -506 or mycophenolic acid.

23, the compound has the formula: %formula%) : 3C; selected from the group consisting of a lβ1.4GlcNAcβ1.3, R2 is β 1.3GaI, β1.2Man or (Xl, 6GalNAc, and hand a is 0 or 1, and X is protein or lipid) 22. The composition according to claim 21.

24, a glycoprotein in which X has a molecular weight of from 40.000 to about 250.000 Daltons; 24. The composition according to claim 23, wherein the composition is a woody substance.

25, X is a glycolipid having a molecular weight of about 600 to about 4,000 Daltons; 24. The composition according to claim 23.

26, the compound has the formula: %formula% X is -H5 lower alkyl (C, -CI), arylalkyl, hydroxy (C, -C,)alkyl or (au-08)alkoxy-(C.

−〇,) is alkyl) A composition according to claim 2o, comprising a component having:

27. said selectin receptor is expressed on vascular endothelial cells or platelets; A composition according to claim 20.

28, a pharmaceutically acceptable carrier, and selectively binds to a selectin receptor comprising a compound having the formula:%formula%) R3 does not contain sialic acid, Rs(Ra)C(CotH)-1 or sialic acid. It is a trisaccharide that R1 and R4 are the same or different and -H2-CI-C8 a alkyl, -hydroxyl substituted C1-C8 alkyl, -aryl substituted CI-CB a alkyl, or -alkoxy substituted Cl-C8 alkyl, and R2 is β1.3Ga 1. irl, 2Man, and β1.6GalNac. ri, and a is 0 or 1, and X is -H5 lower alkyl (C, -C,), hydroxyalkyl (CI-C6), Alkoxy (C, -C,), alkyl (CI-Cs>, -aryl, -alkyl (selected from the group consisting of aryl and -arylalkyl) A pharmaceutical composition comprising:

29. The above compound is 2: NeuA, cα2,3Galβ1.4 (Fuccrl, 3)GlcNAc-( R), , NeuGcα2,3Ga lβl, 4 (Fuc+rl.

3) GI cNAc-(R)- and NeuGctx2.3C; alβ1゜4 GIcNAcβL, 3Galβ1,4 (Fuc+r1.3)GlcNAc-( R), (where R is β1,3Gal, β1.2Man or α1,6Nac, and a is O or 1) 29. The composition of claim 28.

30, a pharmaceutically acceptable carrier, and selectively binds to a selectin receptor comprising a compound having two or more repeating units capable of , said repeating unit comprises a selectin-binding component and is bound by a linker moiety. are chained, each repeating unit having the formula: %formula%) R3 is composed of sialic acid, an oligosaccharide comprising sialic acid, and a group having the formula: a member selected from the group R3 and R4 are individually the same, but and -H,,C,-C,alkyl,hydroxyCC,-C,a (alkyl), aryl-(C, -c, alkyl) and (CI-CS alkoxy) -(C, -C, alkyl), or R3 and R1 together are 4 ~ forms an 8-membered ring, and R8 is β1,3Gal, txi2Man or cr l, 6GalNac, and a is O or 1, and X is -H1 lower alkyl CC+ Cs), arylalkyl, human o+Cs (CI Co) Furkyl or (CI CI)77L/Dxy'-(CI-08) Al kill) A pharmaceutical composition comprising:

31, the linker component has the formula: n and m are the same or different and are integers from 2 to 12; Y +, t O or S, and W is O4S or NH) 31. The composition of claim 30.

32, the linker part is a 5- to 14-membered ring having two substituents, and each substituent is the formula: %formula%) and the substituents are in a trans or trans relationship. Composition.

33, the substituent is in a configuration of 1.2 to 1, (p/2)+1, where p is 5 to 33. A composition according to claim 32, wherein the number is an integer of 4 and corresponds to a ring size.

34, a pharmaceutically acceptable carrier, and two nitrogen atoms and two selecti a heterocyclic compound having bonding moieties, each moiety being linked to one of the nitrogen atoms. chained, and the expression: %expression%() R1 contains sialic acid, Rj(R,)C(CoffiH)-1 or sialic acid It is a trisaccharide consisting of R1 and R4 are the same or different and -H1-C1-C8 a alkyl, -hydroxyl substituted C1-C8 alkyl, -aryl substituted ci-cs alkyl, -hydroxyl substituted C1-C8 alkyl, -aryl substituted ci-cs alkyl, or -alkoxy substituted Cl-C8 alkyl, and R2 is β1.3GaI, al, 2Man, or α1.6GalNac ,and a is O or 1) A pharmaceutical composition comprising:

35. Claim 34, wherein the heterocyclic compound is piperazine or homopiperazine. The composition described in .

36, a pharmaceutically acceptable carrier, and NeuAc+r2,3Ga Isl , 4 (Fucαl, 3)GIcNAc-(R)Il-1NeuGca2.3G alβ1,4 (Fuccrl.

3) Gl cNAc-(R)-1 and NeuGcα2,3Galβ1.4Gl cNAcβ1.3Calβ1.4 (Fucαl, 3)GlcNAc (R)- (here R is β1.3Cal, α1, 2Man, or α1.6Nac, and al, tO or I) an amino acid linked to a selectin-binding oligosaccharide component selected from the group consisting of A medical composition comprising:

37. The above amino acids are lysine, homolysine, orthinine, diaminobutyric acid, aspar 37. The composition of claim 36, which is ragin or diaminopropionic acid.

38. The amino acid according to claim 36, wherein the amino acid is incorporated into an oligopeptide. composition.

39, the oligopeptide consists of one or more of the following: lysine; Homolysine, ortinine, diaminobutyric acid, asparagine or diaminopropio 3. The composition according to claim 3.

40. The oligopeptide consists of one or more of the following: alanine , tyrosine or radioactive tyrosine iodide, the composition according to claim 39@! J.

41, the oligopeptide is arranged in a direction from the N-terminus to the C-terminus (where R1 and R2 is the same or different and is any amino acid residue, and 39. The composition of claim 38, wherein L is an oligosaccharide component.

42, a pharmaceutically acceptable carrier, and a selectin cell surface receptor. Immunoglobulins that can selectively bind to recognized oligodecal ligands wherein the immunoglobulin is present in an amount sufficient to treat the inflammatory condition. A pharmaceutical composition.

43, said ligand has the formula: %formula% 43. The composition of claim 42, selected from the group consisting of.

44. The composition of claim 42, wherein the oligosaccharide component is expressed by leukocytes. .

45. The selectin receptor is expressed by vascular endothelial cells or platelets. 43. The composition of claim 42.

46, the selectin receptor is ELAM-1 or GMP-140; 43. A composition according to claim 42.

47. The composition of claim 42, wherein said composition is in unit dosage form.

48, a pharmaceutically acceptable carrier, and selectively binds to a selectin receptor comprising a compound comprising a component having the formula: %formula%) Kyl-Araαl, 3 and (R,5)-5-aryl-Aracrl, 3. ri, and R8 is 1.3βGa l, 1,2αMan and l,6αGa1NAc, and a is O or 1, and X is -H2 lower alkyl (C, -C,), arylalkyl, human 0 * cy ( C+ Cm) 7) Lt kill, mata is (C+ -Cs) alkoxy (C, - C,) is alkyl) A pharmaceutical composition comprising:

49. The composition of claim 48, wherein the compound is a biological molecule.

50. Selectin receptor in which the component is expressed on vascular endothelial cells or platelets 49. The composition of claim 48, wherein the composition binds to -.

51, the selectin receptor is ELAM-1 or GMP-140; 49. A composition according to claim 48.

52. A pharmaceutically acceptable carrier and selectivity in a therapeutically effective dose to the patient. administering a pharmaceutical composition comprising a compound that selectively binds to a receptor; A method of inhibiting selectin-mediated cell-cell adhesion in a patient, comprising:

53. the compound comprises a component that selectively binds to selectin receptors; , the said components have the formula: %formula%) R1 does not contain sialic acid, R, (R,)C(CotH)-2 or sialic acid. It is a trisaccharide that R1 and R4 are the same or different and -H1-C1-(l a alkyl, -hydroxyl monosubstituted C1-C8 alkyl, -aryl substituted C1, -C8 alkyl, or -alkoxy substituted C1-CB alkyl, and R2 is β1.3Gal, αl, 2Man, or αl, 6GalNac, and a is O or 1, and X is -H1 lower alkyl (C, -C,), arylalkyl, hydroxy (C1 Cs) alkyl, or (C, -Ce)alkoxy-cc, -cs > alkyl be) 53. The method of claim 52, comprising:

54, the compound is (C, -C,)alkyl, (C, -C,)alcoquino-(C 53. The method of claim 52, wherein tCt)alkyl.

55. The method of claim 52, wherein the cell-cell adhesion is associated with an inflammatory condition.

56. The method of claim 55, wherein the inflammatory condition is septicemia.

57. The inflammatory condition is 2. respiratory distress syndrome or wound-associated sepsis. 56. The method of claim 55.

58. The method of claim 52, wherein said cell-cell adhesion is associated with metastasis.

59. The selectin receptor promotes the attachment of leukocytes, monocytes or neutrophils to endothelial cells. 53. The method of claim 52, further comprising:

60, the selectin receptor is ELAM-1 or GMP-140 53. The method of claim 52.

61. The method of claim 52, wherein the compound is encapsulated within a liposome. .

62. The method of claim 52, wherein the compound is a polysaccharide.

63 selectively binds to selectin receptors in therapeutically effective doses to the patient. comprising administering a compound, said compound having the formula %formula%) R3 is sialic acid, R3(R,)C(COz　)()-, or a triester consisting of sialic acid sugar, R3 and R1 are the same or different and -H1-ct-cs a alkyl, -hydroxyl substituted C1-C8 alkyl, -aryl substituted CL-CB alkyl or monoalkoxy-substituted Cl-08 alkyl, and Rz is β1.3Gal, (rl, 2MaH or αl, 6GalNac, and a is O or 1, and X is -H1 lower alkyl (C, -CI), arylalkyl, hydroxy (C, -cm>alkyl or (C, -C,)alkoxy-(C.

-C1) is alkyl) of inflammatory diseases mediated by selectin receptors in patients with How to process.

Claim 6, wherein X is an oligonucleotide, an oligopeptide, a protein or a lipid. The method described in 3.

65, the selectin receptor is ELAM-1 or GMP-140; 64. The method of claim 63.

66. Process: A test compound is contacted with a selectin receptor and an isolated selectin binding agent. Let, and the ability of the test compound to inhibit the binding between the receptor and the selectin binding agent; detect force, Test compounds are measured for their ability to inhibit selectin-mediated cell adhesion comprising How to determine.

67, the binder has an SLχ component or a pseudo-SLX (SLX mimetic) 67. The method of claim 66, comprising:

68. Claim 6, wherein the receptor or binding agent is immobilized on a solid surface. 6. The method described in 6.

69. The method according to claim 66, wherein the test compound is an oligomer or a sugar conjugate. Law.

70, blocking selectin-mediated cell adhesion identified by the method of claim 66. A pharmaceutical composition comprising a compound capable of binding.

71, formula: %formula% (where R is sialic acid) a selectin comprising fucosylating a polysaccharide comprising a sequence having of a compound comprising an oligosaccharide moiety capable of selectively binding to a receptor Production method.

72. The fucosylation step is carried out using fucosyltransferase. 72. The method of claim 71.

73. Claim 72, wherein the group B Streptococcus polysaccharide is a type Ia polysaccharide. the method of.

74. Claim 72, wherein the multi-W class is a group B Streptococcus type II. the method of.

75, linking multiple components that can selectively bind to selectin receptors. comprising chaining together using a car component. Method for manufacturing compounds.

76, the component has the formula: %formula%() R1 is sialic acid, R,, (R,) sugar, R3 and R4 are the same or different and -Hl-CI-08a alkyl, -hydroxyl substituted Cl-C8 alkyl, -aryl substituted Cl-C8 alkyl, -hydroxyl substituted Cl-C8 alkyl, -aryl substituted Cl-C8 alkyl, or -alkoxy substituted c1-C8 alkyl, and a is O or 1) 76. The method of claim 75.

77, the linker component has the formula: n and m are the same or different and are integers from 2 to 12; Y is O or S, and W is 0.3 or NH) 76. The method of claim 75.

78, the linker part is a 5- to 14-membered ring having two substituents, and each substituent is the formula: %formula%) and the a group is in a cis or trans relationship. Method.

79, the substituents are in the configuration 1.2-1, (p/2)+1, where p is 5-14 76. The method of claim 75, wherein the integer is an integer of and corresponds to the size of the ring.

international search report 111+-1-^--1 Pσ/+JS91.104284

Claims (81)

[Claims] 1. selectively binds to pharmaceutically acceptable carriers and selectin receptors comprising a compound comprising a component, said component having the formula: R1-Galβ1,4(Fucα1,3)GlcNAc-(R2)■ (where, R1 is an oligosaccharide or R3-R4-C(CO2H)-, and R3 and R4 are are the same or different, and -H, -C1-C8 alkyl, -hydro xyl C1-C8 alkyl, -aryl C1-C8 alkyl or -alkoxy C1-C8 alkyl, R2 is β1,3Gal, α1,2Man or α1,6GaINaC; do a is 0 or 1) A pharmaceutical composition comprising: 2. 2. The composition of claim 1, wherein the compound is a biological molecule. 3. the biological molecule is an oligosaccharide, an oligopeptide, a protein or a lipid; A composition according to claim 1. 4. The composition according to claim 1, wherein R3 and R4 form a 4- to 8-membered ring. 5. The composition according to claim 4, wherein the 4- to 8-membered ring is a monosaccharide. 6. 6. The composition of claim 5, wherein the monosaccharide is sialic acid. 7. Claim wherein the sialic acid is NeuAcα2,3 or NeuGcα2,3 The composition according to item 6. 8. The oligosaccharide is NeuAcα2,3Galβl,4GlcNAcβl,3 or or NeuGcα2,3Galβl,4GlcNAcβl,3, Claim 1 The composition described in . 9. 2. The composition of claim 1, wherein the compound is a polysaccharide. 10. The oligosaccharide has the formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 10. The composition of claim 9, comprising a repeating unit having: 11. The oligosaccharide has the formula: 10. The composition of claim 9, comprising a repeating unit having: 12. The oligosaccharide has the formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 10. The composition of claim 9, comprising a repeating unit having: 13. The claim is that the polysaccharide is a group B Streptococcus fucosylated type Ia polysaccharide. The composition according to claim 9. 14. The polysaccharide is a group B Streptococcus type II or type III polysaccharide. 10. The composition of claim 9. 15. 10. The polysaccharide of claim 9, wherein the polysaccharide has a molecular weight of about 5,000 to 300,000. Compositions as described. 16. 9. The polysaccharide comprises from about 5 to about 200 fucosylated repeat units. The composition described in . 17. 4. The polysaccharide comprises from about 25 to about 100 fucosylated repeat units. 16. The composition according to 16. 18. 2. The composition of claim 1, wherein the biomolecule is a sphingolipid. 19. 19. The composition of claim 18, wherein the biomolecule is a ganglioside. 20. the selectin receptor is expressed on vascular endothelial cells or platelets; A composition according to claim 1. 21. the selectin receptor is ELAM-1 or GMP-140; A composition according to claim 20. 22. selectively binds to pharmaceutically acceptable carriers and selectin receptors A pharmaceutical composition comprising a ribosome having a compound. 23. 23. The ribosome encapsulates an anti-inflammatory chemotherapeutic agent. Composition of. 24. The anti-inflammatory agent is cyclosporin A, indomethacin, naproxen, FK -506 or mycophenolic acid. 25. The compound has the formula R1-Galβl,4(Fucαl,3)GlcNAc-(R2)■-X(here in R1 is NeuAcα2,3, NeuGcα2,3, NeuAcα2,3Galβ l,4GlcNAcβl,3 and NeuGcα2,3Galβl,4GlcN Acβl,3, R2 is βl,3Gal, αl,2Man or αl,6GalNAc, and a is 0 or 1, and X is protein or lipid) 23. The composition according to claim 22. 26. a glycoprotein in which X has a molecular weight of from 40,000 to about 250,000 Daltons; 26. The composition of claim 25, which is a woody substance. 27. Claim 2, wherein X has a molecular weight of about 600 to about 4,000 Daltons. 5. The composition according to 5. 28. The compound has the formula: NeuAcα2,3Galβl,4(Fucαl,3)GIcNAc-(R)■ -, NeuGcα2,3Galβl,4(Fucαl,3)GlcNAc-(R )■- and NeuGcα2,3Galβl,4GlcNAcβl,3Galβ l,4(Fucαl,3)GlcNAc-(R)■-(where, R is βl,3Gal, αl,2Man or αl,6Nac, and a is 0 or 1) 23. The composition of claim 22, comprising a moiety having: 29. the selectin receptor is expressed on vascular endothelial cells or platelets; A composition according to claim 22. 30. selectively binds to a pharmaceutically acceptable carrier and a selectin receptor. wherein the compound has the formula R1-Galβl,4(Fucαl, 3) GlcNAc-(R2)■-X (where, R1 is an oligosaccharide or R2-R4-C(CO2H)-, and R3 and R4 are are the same or different, and -H, -lower alkyl (C1-C8), -hydroxyl lower alkyl, -arylalkyl or -alkoxyalkyl and R2 is βl,3Gal, αl,2Man or αl,6GalN ac, and a is 0 or 1, and X is -H, -OH, -NH3, -NHR3, -NR3R4, -OR2, O aryl -Oalkylaryl, -Oarylalkyl, -R3, -aryl, -A (selected from the group consisting of arylalkyl and -alkylaryl) A pharmaceutical composition comprising: 31. The compound has the formula: NeuAcα2,3Galβl,4(Fucαl,3)GlcNAc-(R)■ , NeuGcα2,3Galβl,4(Fucα1,3)GlcNAc-(R) ■ and NeuGcα2,3Galβl, 4GlcNAcβl, 3Galβl, 4(Fucαl,3)GlcNAc-(R)■ (here R is βl,3Gal, αl,2Man or αl,6Nac, and a is 0 or 1) 31. The composition of claim 30. 32. selectively binds to a pharmaceutically acceptable carrier and a selectin receptor. comprising a compound having two or more repeating units capable of The repeating units comprise selectin-binding components and are linked by linker moieties. and each repeating unit has the formula: R1-Galβl,4(Fucαl,3)GlcN Ac-(R2)■-X (where, R1 is an oligosaccharide or R3-R4-C(CO2H)-, and R3 and R4 are are the same or different, and -H, -C1-C8 alkyl, -hydro xyl C1-C8 alkyl, -aryl C1-C8 alkyl or -alkoxy C1-C8 alkyl, R2 is βl,3Gal, αl,2Man or αl,6GalNac; do a is 0 or 1, and X is a linker component) A pharmaceutical composition comprising: 33. The linker component has the formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼or▲There are mathematical formulas, chemical formulas, tables, etc.▼ (here, n and m are the same or different and are integers from 2 to 12; Y is O or S, and W is O, S or NH) 33. The composition of claim 32. 34. The linker moiety is a 5- to 14-membered ring having two substituents, and each substituent is the formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ here (Y is O or S) and the substituents are in a cis or trans relationship. Composition. 35. The substituents are in a configuration of 1,2-1, (p/2)+1, where p is 5-1. 35. The composition of claim 34, which is an integer of 14 and corresponds to the size of the ring. 36. a pharmaceutically acceptable carrier and two nitrogen atoms and two selectins comprising a heterocyclic compound having bonding moieties, each moiety linked to one of the nitrogen atoms. and the formula: R1-Galβl,4(Fucαl,3)GlcNAc -(R2)■(Here, R1 is an oligosaccharide or R3-R4-C(CO2H)-, and R3 and R4 are are the same or different, and -H, -C1-C8 alkyl, -hydro xyl C1-C8 alkyl, -aryl C1-C8 alkyl or -alkoxy C1-C8 alkyl, R2 is βl,3Gal, αl,2Man or αl,6GalNac; do a is 0 or 1) A pharmaceutical composition comprising: 37. 36. The heterocyclic compound is piperazine or homopiperazine. The composition described in . 38. A pharmaceutically acceptable carrier and: NeuAcα2,3Galβl,4( Fucαl, 3) GlcNAc-(R)■-, NeuGcα2,3Galβl, 4(Fucαl,3)GlcNAc-(R)■- and NeuGcα2,3Ga lβl,4GlcNAcβl,3Galβl,4(Fucαl, 3)GlcNA c-(R)■ (where R is βl,3Gal, αl,2Man or αl,6Nac, and a is 0 or 1) A chain of amino acids of a selectin-binding oligosaccharide component selected from the group consisting of A pharmaceutical composition comprising. 39. The amino acids mentioned above include lysine, homolysine, orthinine, diaminobutyric acid, and aspart. 39. The composition of claim 38, which is ragin or diaminopropionic acid. 40. 39. The amino acid according to claim 38, wherein the amino acid is incorporated into an oligopeptide. composition. 41. The oligopeptide comprises one or more of the following: homolysine, ortinine, diaminobutyric acid, asparagine or diaminopropylene. Pionic acid, the composition of claim 40. 42. The oligopeptide comprises one or more of the following: 42. The composition of claim 41, wherein tyrosine, tyrosine or radioiodide tyrosine. 43. In the direction from the N-terminus to the C-terminus, the oligopeptide There are formulas, tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (where R1 and R2 are the same or different and any amino acid and L is an oligosaccharide moiety. composition. 44. recognized by pharmaceutically acceptable carriers and selectin cell surface receptors. immunoglobulins that can selectively bind to the oligosaccharide ligands identified wherein said immunoglobulin is present in a sufficient amount to treat an inflammatory condition. A pharmaceutical composition. 45. The ligand has the formula: NeuAcα2,30alβl,4(Fucαl,3)GlcNAc-(R)■ -, NeuGcα2,3Galβl,4(Fucαl,3)GlcNAc-(R )■- and NeuGcα2,3Galβl,4GlcNAcβl,3Galβ l,4(Fucαl,3)GlcNAc-(R)■-(here R is βl,3Gal, αl,2Man or αl,6Nac, and a is 0 or 1) 45. The composition of claim 44, wherein the composition is selected from the group consisting of: 46. 45. The composition of claim 44, wherein the oligosaccharide moiety is expressed by leukocytes. . 47. the selectin receptor is expressed by vascular endothelial cells or platelets 45. The composition of claim 44. 48. the selectin receptor is ELAM-1 or GMP-140; 45. The composition of claim 44. 49. 45. The composition of claim 44, wherein the composition is in unit dosage form. 50. selectively binds to a pharmaceutically acceptable carrier and a selectin receptor. wherein said component has the formula: R1-Galβ1,4(R 2) GIcNAc-(R3)■-(where, R1 is NeuAcα2,3, NeuGcα2,3, NeuAcα2,3Galβ l,4GlcNAcβl,3 or NeuGcα2,3Galβl,4GlcN Acβl,3, R2 is Fucαl,3, Araαl,3, (R,S)-5 -Alkyl-Araal, 3 and (R,S)-5-aryl-Araal, 3 is, and R3 is 1,3βGal, 1,2αMan and 1,6αGalNAc; do a is 0 or 1) A pharmaceutical composition comprising: 51. 52. The composition of claim 51, wherein the compound is a biological molecule. 52. a selectin receptor in which the component is expressed on vascular endothelial cells or platelets; 52. The composition of claim 51, wherein the composition binds to -. 53. the selectin receptor is ELAM-1 or GMP-140; 52. The composition of claim 51. 54. a pharmaceutically acceptable carrier and selectivity in a therapeutically effective dose to the patient; administering a pharmaceutical composition comprising a compound that selectively binds to a receptor; A method of inhibiting selectin-mediated cell-cell adhesion in a patient, comprising: 55. The compound comprises a component that selectively binds to a selectin receptor. , the said components have the formula: R1-Galβl,4(Fucαl,3)GlcNAc-(R2)■ (where, R1 is an oligosaccharide or R3-R4-C(CO2H)-, and R3 and R4 are are the same or different, and -H, -lower alkyl (C1-C8), -hydroxyl lower alkyl, -arylalkyl or -alkoxyalkyl and R2 is βl,3Gal, αl,2Man or αl,6GalN ac, and a is 0 or 1) 56. The method of claim 55. 56. 56. The method of claim 55, wherein the composition is a biomolecule. 57. 56. The method of claim 55, wherein the cell-cell adhesion is associated with an inflammatory condition. 58. 59. The method of claim 58, wherein the inflammatory condition is septic shock. 59. The inflammatory conditions are acute respiratory distress syndrome and wound-related sepsis. 59. The method of claim 58. 60. 56. The method of claim 55, wherein said cell-cell adhesion is associated with metastasis. 61. The selectin receptors promote the attachment of leukocytes, monocytes, or neutrophils to endothelial cells. 56. The method of claim 55, wherein the method mediates the transfer. 62. the selectin receptor is ELAM-1 or GMP-140; 56. The composition of claim 55. 63. 56. The method of claim 55, wherein the compound is encapsulated within a ribosome. . 64. 56. The method of claim 55, wherein the compound is a polysaccharide. 65. selectively binds to selectin receptors in therapeutically effective doses to patients comprising administering a compound having the formula: R1-Galβl,4( Fucαl, 3) GlcNAc-(R2)■-X (where, R1 is an oligosaccharide or R3-R4-C(CO2H)-, and R2 and R4 are are the same or different, and -H, -lower alkyl (C1-C8), -hydroxyl lower alkyl, -arylalkyl or -alkoxyalkyl and R2 is βl,3Gal, αl,2Man or αl,6GalN ac, and a is 0 or 1, and X is a biological molecule) inflammatory disease hypersensitivity mediated by selectin receptors in patients with How to treat the problem. 66. Claim 6, wherein X is an oligosaccharide, oligopeptide, protein or lipid. 6. The method described in 6. 67. the selectin receptor is ELAM-1 or GMP-140; 67. The composition of claim 66. 68. Process: A test compound is contacted with a selectin receptor and an isolated selectin binding agent. Let, and the ability of the test compound to inhibit the binding between the receptor and the selectin binding agent; detect force, Test compounds are measured for their ability to inhibit selectin-mediated cell adhesion comprising How to determine. 69. The binder contains an SLX component or a pseudo SLX (SLX mimetic). 70. The method of claim 69. 70. 6. The receptor or binding agent is immobilized on a solid surface. 9. 71. 70. The method according to claim 69, wherein the test compound is an oligosaccharide or a sugar conjugate. Law. 72. Blocking selectin-mediated cell adhesion identified by the method of claim 69 A pharmaceutical composition comprising a compound capable of binding. 73. formula: R-Galβl,4GlcNAc- (Here R is sialic acid) selectin receptor, which consists of fucosylating a polysaccharide comprising a sequence with Preparation of Compounds Comprising Oligosaccharide Moieties That Can Selectively Bind to Proteins how to. 74. The fucosylation step is carried out using αl,3 fucosyltransferase. 75. The method of claim 74, wherein the method is performed. 75. Claim 74, wherein the Group B Streptococcus polysaccharide is a type Ia polysaccharide. the method of. 76. The claim is that the polysaccharide is of group B Streptococcus type II or III. The method according to claim 74. 77. Links multiple moieties that can selectively bind to selectin receptors a compound comprising said components, consisting of chaining them together using car components; How to prepare. 78. The above part is the formula: R1-Galβl,4(Fucαl,3)GlcNAc-(R2)■ (where, R1 is an oligosaccharide or R3-R4-C(CO2H)-, and R3 and R4 are are the same or different, and -H, -C1-C8 alkyl, -hydro xylC1-C8 alkyl, -arylC1-C8 alkyl or -alkoxy C1-C8 alkyl, R2 is βl, 3Gal, αl. 2Man or αl,6GalNac, and its do a is 0 or 1) 79. The method of claim 78, comprising: 79. The linker component has the formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼or▲There are mathematical formulas, chemical formulas, tables, etc.▼ (here n and m are the same or different and are integers from 2 to 12; Y is O or S, and W is O, S or NH) 79. The composition of claim 78. 80. The linker component is a 5- to 14-membered ring having two substituents, and each substituent The group has the formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ here (Y is O or S) and the substituents are in a cis or trans relationship. Composition. 81. The substituents are in a configuration of 1,2-1, (p/2)+1, where p is 5-1. 79. The composition of claim 78, which is an integer of 14 and corresponds to the size of the ring.