JPH07504222A - Suppression of cell adhesion by chemically defined oligosaccharides, derivatives and mimetics of the oligosaccharides, and antibodies against the oligosaccharides - 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] Chemically defined oligosaccharides, derivatives and mimetics of said oligosaccharides and Cross-reference of applications related to inhibition of cell adhesion by antibodies This application is based on U.S. Patent Application No. 071575539 filed August 30, 1990 ( This is a partial continuation application (abandonment). Pending U.S. patent filed July 2, 1991 This is a continuation-in-part of Application No. 07/724.983. November 12, 1991 This is a continuation-in-part of pending U.S. patent application Ser. No. 07/789,969. U.S. Patent Application No. 07/836°9, filed February 19, 1992; This is a partial continuation application of No. 78. Pending U.S. patent application filed September 25, 1992 This is a continuation-in-part of Application No. 07/950,720.

All five applications mentioned above are incorporated by reference into this disclosure.

Technical field The invention generally relates to adhesion of tumor cells or inflammatory leukocytes to specific cell types. Concerning the suppression of metastasis and invasion of tumor cells and inflammatory processes based on the suppression of inflammatory processes.

More specifically, the present invention provides tumor-associated carbohydrate antigens, leukocyte-associated carbohydrate antigens, These oligosaccharide derivatives, tumor-associated carbohydrate antigens and leukocyte-associated carbohydrate antigens Inhibition of the above by using antibodies against tumor-associated carbohydrate antigens as well as tumor-associated carbohydrate antigens. related.

Background of the invention Despite huge investments in financial and human resources, cancer remains one of the leading causes of death. It is. Current cancer treatments cure only about half of all patients with malignant tumors. It doesn't heal. Metastasis is the major cause of death in most human malignant tumors.

Metastasis is the formation of secondary tumor colonies at one or more distant sites. The metastasis is It is a multistep process where tumor invasion is the first step.

Tumor cells locally invade host tissue barriers such as the epithelial basement membrane and invade the interstitial stroma. Once there, the tumor cells access blood vessels (or lymphatic vessels) and metastasize further. do. After invading the endothelial layer of the vessel wall, circulating tumor cells are expelled by the circulating flow and endothelial cells Precapillary venules of target organs by adhering to the luminal surface or exposed basement membrane Stay in. The tumor cells invade the blood vessel wall again and enter the parenchyma of the organ. lastly, The infiltrating tumor cells grow in different tissues than the one in which the tumor originated.

In most human malignant tumors, distant metastases are too small to be detected during treatment of the primary tumor. I can't get it out. In addition, widespread initial onset of metastatic colonies usually leads to Occurs before clinical symptoms of a transmissible disease become apparent. size of metastases, patient age, The anatomical location and heterogeneous composition all impede surgical removal of the tumor and lead to metastasis. The concentration of anticancer drugs that can reach the sexual colonies is limited.

Inhibiting the metastatic potential of tumor cells, as current methods of treating and preventing metastases are difficult. There is a need in the art for improved methods and compositions that allow for.

The present invention meets these needs and further provides other related advantages.

On the other hand, there is a common mechanism between the inflammatory process and the initiation of metastasis. For example, both The microvascular processes of cells (white blood cells in the former case and tumor cells in the latter case) Adhesion to endothelial cells triggers white blood cells or tumor cells to cross the endothelial cells and assemble. Move to the weaving space. Both processes are enhanced by activated platelets. Also, Both processes are linked to tumor-associated carbohydrate antigens (TACA) or leukocyte-associated carbohydrates. It is strongly mediated by specific types of carbohydrates such as antigens (LACA). Some kind of TACA shares a structure with LACA.

The present invention provides, for example, cell adhesion mediated by TACA or LACA, e.g. It is related to and based on the inhibition using antibodies of the human body.

Summary of the invention Briefly, according to the present invention, carbohydrate structures or their antibodies can be used to treat tumors. Compositions and methods for inhibiting tumor cell metastasis and invasion based on blocking cell adhesion are disclosed. provided. Further, according to the present invention, whitening by carbohydrate structures or their antibodies is achieved. Compositions and methods are provided for inhibiting leukocyte inflammation based on blocking blood cell adhesion.

The principles of this technique are: (a) carbohydrate-carbohydrate interactions; (b) carbohydrate-separate interactions; or (C) blocking both of them. for example , l) Model using mouse melanoma B16 mutant with high and low metastatic potential In multiple experiments, the highly metastatic mutants BL6 and FIO were compared to the hypometastatic or non-metastatic mutants. The variant expresses more GM3 than F1 or Wa4. Highly metastatic to endothelial cells The adhesiveness of the mutant is greater than that of the low metastatic mutant, and this adhesiveness is - Lactocyto, GM3 or LacCer (each in liposomes) or other LacCer Inhibited by kutocyto derivatives. Saccharides and derivatives are also associated with B16 melanoma metastasis suppress. This is due to (a) above, that is, inhibition of carbohydrate-carbohydrate interactions. This is an example.

ij) Regarding human cancer, the -order tumor is H/Le'/Leb (monoclonal antibody MI A-15-5), Sialosyl Tn (antibody TKH2) ) or sialosyl-Lef (monoclonal antibody FH6,5NH Patients who express known tumor-associated carbohydrate antigens such as - Do patients whose tumors do not express these antigens or express them weakly have a much lower survival rate than patients? It was.

1ii) These tumor-associated carbohydrate antigens (GM in mouse melanoma models) 3 and H/Le'/Le', Sialosyl-Lex or Sia in human tumors Rosyl-Tn) is a substance recognized by target cells, especially platelets or endothelial cells. is an adhesion molecule. The above suppresses the combination approach, i.e. (a) and (b) This is an example of a method.

iv) In the interaction of tumor cells with endothelial cells and platelets, activated endothelial cells and activated LECCAM (selectin), ELAM-1 or GMP-140 intervenes. Sialosyl-Le' antigens are associated with these LECCAM It is known that it is recognized by The above is a carbohydrate-selectin interaction This is an example of (b) affecting.

V) Expression on platelets or endothelial cells of thrombin, ADP or (AMP) GMP-140 induced by ball ester inhibits platelet-tumor cell interaction plays an important role in tumor cell metastasis. recognized by selectins The epitope is sialosyl-Le' (Pol) ey et al., Proc, Na t1. Acad.

Sci, 88:6224.1991); Le” (also known as Monosiaron Le’T), Monosia Rosyl-Le’ll (positional isomer of sialosyl-Le’) and Zinaron-L It was found that GMP-140 also recognizes “e”. It binds better to sialosyl Le'' than to allosyl Le'.

The above is another example of process (b).

vj) Expression on endothelial cells is interleukin-1, TGF-β, TNF-α or ELAM-1 induced by lipopolysaccharide is induced by endothelial cells-leukocytes and endothelial cells. - plays an important role in tumor cell interactions, mediates tumor cell metastasis, and endothelial cells - mediate leukocyte interactions and transmigration of leukocytes and tumor cells through endothelial cells. Ru. The epitope recognized by the above selection was previously Aron Le' (Phillips et al., 5science250:1130. 1990; Berg et al., J. Biol. Chem. 266:14869.199 1. Takayama et al., Biochem, Biophys, Res, Commun, 179 13, 1991), but selectin epitopes have also been identified, particularly In a dynamic flow system, monosialosyl=Le' II and zinearon Le' It was found to be an internally sialylated secondary fuco/lylated l-type or type-2 chain. deer While the binding phenomenon is viprant. Under static or low shear stress dynamic conditions , ELAM-1 (also known as E-selectin) mainly acts on SLe' and α2→3 soarylation of SLe” etc. and αl→3 or α of SLe’ and SLe” etc. Recognizes 1→4 fucosylated carbohydrates. However, moderate to high shear stress dynamic conditions Below, the component expressed by equation (1) or (i I) such as Le”/S L e The progeny (see, e.g., Figure 20) present a high affinity binding site for E-selectin. plays an important role in providing This role is especially pronounced under high shear stress conditions. It is.

vii) Human colon tumor cells showing differential expression for metastatic potential in nude mice The cells showed a close correlation with the expression of Noaron Le'. That is, it has high metastatic properties. cells expressing high levels of sialosyl-Le'' and vice versa.

v i i 1) Adhesion of E-selectin expressing cells to SLe' ' is greatly enhanced when mixed with liposomes containing various amounts of Le'.

Therefore, it has not only the hybrid SLe"/Le" but also SLe" and Le'. A greatly enhanced adhesion is observed in the mixed glycoliposomes.

ix) Human endothelial cells are characterized by high expression of H (Fucα1→2Ga1), And many human cancers are confirmed by monoclonal antibody MIA-15-5. It is characterized by expressing Le', H or Leb. Interaction between H and Le’ or H The interaction with H has been clearly confirmed and therefore it is possible to generate H/Le'/Le'. These human tumors presently show that H May adhere to expressing endothelial cells. The above has implications for carbohydrate-carbohydrate interactions. - Example of process (a) that has an impact.

x) Monoclonal antibody MIA-15-5 against H/Le'/Le' inhibited lung metastasis of highly metastatic FIO and BL6 mutant cells in mice. moreover , monoclonal to GinAronru Le' and Monosialosilu Le'TI Antibody FH7 is used to adhere human cancer cells expressing the above antigen in a dynamic flow system. was suppressed.

As a result of the above and other various considerations and studies, the present invention provides the following: It will be done.

a) GM3, I], Le' as shown in structures 1-14 in Example 3 , Le”, monosialosyl-Le’ (SLe’), Le’, Le’ , Le”/SLe” hybrid (Structure I in Figure 20), etc. Saccharides, monosialosyl-Le'I (SLe''), monosialosyl-Le'll , sialosyl Tn, lactonol and other structures. Compositions and methods for suppressing tumor cell metastasis due to hydrate antigen-mediated tumor cell adhesion Law.

b) Le’ / SLe” hybrid or related components such as Le’ and SLe” can be a suitable mixture, especially under high shear stress conditions in dynamic flow systems. Compounds such as those shown in Figure 20 that provide high affinity adhesive binding sites. However, Therefore, such compounds may inhibit E-select of tumor cells or leukocytes against endothelial cells. Block Chin-mediated adhesion.

C) Oligosaccharide derivatives based on the above structures, which are bound to suitable carriers. sugar derivatives.

d) By appropriately changing the sugar structure, oligosaccharide-lectin (selectin; LEccA M) or a better block for tumor cell adhesion based on oligosaccharide-oligosaccharide interactions. An oligosaccharide derivative that exhibits locking activity.

e) an oligo comprising and representing a tumor-associated carbohydrate antigen; Endothelial cells, platelets or target cells can also be detected by using antibodies that recognize sugars. It is possible to suppress tumor cell adhesion to and suppress metastasis.

e) a combination of antibodies that recognize oligosaccharides involved in cell adhesion and representing tumor-associated antigens; Use of wase.

That is, according to one aspect of the present invention, tumor cell metastasis and inflammation within a biological sample are suppressed. A method is provided to control the situation. This method inhibits the metastasis of biological samples, said formulations (a) Tumor-associated carbohydrate antigens (or leukocyte-associated carbohydrate antigens) with unique prognostic significance; hydrate antigen), (b) an antibody that specifically binds to the above antigen, (c) an origin of the above antigen. a gosaccharide moiety, (d) a complex of the above antigens or oligosaccharides, and (e) a tumor-associated carbohydrate antibody. at least one selected from the group consisting of mimics of the antigen (or leukocyte-associated carbohydrate antigen); Also comprises incubation with a drug. appropriate biology Targeted samples include cell cultures and biological fluids.

According to another aspect of the invention, the method of suppressing tumor cell metastasis or inflammation in warm-blooded animals A method is provided. This method inhibits tumor cell metastasis and inflammation in warm-blooded animals. Inhibits (a) tumor-associated carbohydrate antigens (or leukocyte-associated carbohydrate antigen), (b) an antibody that specifically binds to the above antigen, (c) an antibody of the above antigen. an oligosaccharide component, (d) a complex of the above antigen or oligosaccharide, and (e) a tumor-associated carbohydrate. an effective mimetic of the antigen (or leukocyte-associated carbohydrate antigen); the method comprising administering an amount of at least one drug.

In a related aspect, the present invention provides methods for increasing the in vivo half-life of oligosaccharide components. A wide variety of glycoconjugates are provided. These complexes convert oligosaccharides into polyethylene It is bound to lene glycol.

Additional oligosaccharide components used within the methods and compositions of the invention include Tose, lacto-N-tetrose, methyl β-D-lactone and phenyl β -D-thiolactone is included. Oligosaccharide components may be used individually or May be used in combination.

Furthermore, according to the present invention, GM that causes metastasis at the tumor site and an inflammatory response at a certain site Various methods of inhibiting P-140-mediated or ELAM-1-mediated cell aggregation or adhesion provided.

One such method is GMP-140-mediated or ELAM-1 within biologics. In a manner that inhibits intervening cell aggregation or adhesion, this method (a) Le' and SLe' (structure l in Figure 20, branch I (b) hybrid sugar molecules such as those comprising type I chains); (b) Le'' and S L A mixture of components of the hybrid sugar of (a) such as e'; (C) a monoalone; -Le' I, Le', Le', Monosialosyl-Le' II, ZinAronru -Le' or sialosyl Le'; (d) Hybrids such as Le'/SLe' Antibodies that specifically bind to sugars or their components: (e) Mononoalone Le '　I, Le', Le', Mononoaronru Le'll, Ginarosilou L Antibody that specifically binds to e' or sialosyl Le'; (f) Le'/SLe ', Monosialosyl-Le' I, Le', Le', Mononoalon Le' hybrid saccharide oligosaccharides such as ll, shisoallotru Le"or sialosyl Le" Gosaccharide component; (g), SLe'/Le', mononoalone Le' 1. Le’ , Le', Mono-a-ron-Le'll, Disiaron-LeA or Sialo Complexes of hybrid saccharides such as "Sil Le" or complexes of oligosaccharide components: and ( h) Le”　　　　/SLe’　　　　 Mononoaronru Le”　I, Le″, Le’　, Monosialon Le'll, gin-allosil Le'or sialosyl Le, etc. at least one drug selected from the group consisting of hybrid saccharide mimetics; The method also includes incubation.

Another method involves GMP-140-mediated activation of tumor cells or sites of inflammation in warm-blooded animals. or inhibit ELAM-1-mediated cell aggregation or adhesion, thereby inhibiting translocation at the above sites. infection or inflammation is reduced. This method is useful for tumors in warm-blooded animals versus mixed-breed animals. (a) Hybrids such as SLe'/Le' that reduce metastasis or inflammation at cellular sites Sugar 1 (b) Mixing of components of hybrid sugar (a) such as L e ’ and SLe ’ (c) Monosialosyl-Le'l, Le', Le'', monosialosyl -Le’ll, Jin Aron Le” or Sialosil Le’; (d) Le’ /SLe”4 hybrid sugar, monoalone Le’ 1.Le’, Le ’, Monoaron Le’ll, Nsialosi Le’ or Sialosil Le (e) an antibody that specifically binds to Le', SLe', Le" or S Mixture of antibodies against components of hybrid sugar such as Le'; (f) Le'/S Hybrid sugars such as Le'', monosialon Le''T, Le'', Le', Mo Nosialosyl-Le" II, di-allosyl-Le' or sialosyl Le8 Ligosaccharide component; (g) Hybrid sugars such as Le"/SLe", monoalone -Le″l5Le″, Le’, monosialosyl-Le”II, nosialonru A complex of Le' or sialosyl Le' or a complex of oligosaccharide components: and (h) Hybrid sugars such as Le” / SLe”, monosialosyl-Le’ L Le’ , Le', monosialosyl-Le'll, gin-aron-le' or sialo The effectiveness of at least one drug selected from the group consisting of mimetics of Sil Le''' comprising administering an amount of

According to the present invention, GMP-140-mediated or inhibits inflammation at these sites by suppressing ELAM-1-mediated cell aggregation or adhesion. A method is provided for reducing sex. This method is used to treat warm-blooded animals with warm-blooded animals. Sugars that are components of hybrid sugars (a) such as SLe' that reduce inflammatory properties at the site of disease. Suitable mixture: (C) Monosialosyl-Le'L Le', Le', Mo Nosialonol Le'II, disialosyl LeI or sialosyl Le8; ( d) Hybrid sugars such as Le/S L e', monosialosyl-Le' L Le’, Le’, monosialosyl-Le”lI, ginarosil-1, An antibody that specifically binds to e' or sialosyl Le'''; (e) especially Le'', Mixture of antibodies against components of hybrid sugars such as SLe', Le' and SLe' (f) Hybrid sugars such as SLe'/Le', monosialosyl-Le' L Le', Le'', Monoaronru Le'' II, Ginarosilou L Oligosaccharide component of e' or sialosyl Le': (g) SLe'/Le8 etc. Ibrid sugar, monosialosyl-Le'I, Le', Le', monosialo Sil=Le'li Shisoallosil Le' or sialosyl I-e x complex or is a complex of oligosaccharide components; and (h) hybrid sugars such as Le'/SLe' , monosialocyl Le “1, Le”, Le’, monosialosyl-Le’ ll, from the group consisting of mimetics of shisoallosyl-Le" or sialosyl-Le" administering an effective amount of at least one selected drug.

According to another aspect of the invention, (A) a TA likely to be a target of GMP-140; A fluorescent probe comprising fluorescent plastic beads coated with a CA epitope. and (B) incubating the fluorescent probe with a suspension of platelets. (C) measuring the degree of binding of the fluorescent probe to platelets; Tumor-associated carbohydrates on the lectin activity of GMP-140 comprising the step of A method for identifying an epitope of a substance antigen (TACA) is provided.

These and other aspects of the invention will be understood by reference to the following detailed description and accompanying drawings. It will become clearer.

Brief description of the drawing Figure 1 shows the effects of methyl β-D-rays on the number and size of lung colonies of BL6 cells. 1 is a graph showing the influence of chthond or methyl β-D-thiolactosite. BL6 Cells were grown in control medium (0,1M methyl β-D-lactocytate (rMe-β-lactocytate). ) or 0,1 M phenyl β-D-thiolactone (rphe-β-8-lactone). Pre-incubation was carried out with Toshito')). 20000 cells C57B+ Mice were injected intravenously. The number of lung colonies was counted on day 21 and shown in each bar. They were classified based on the diameter (>1 mm vs. <1 mm). The number of colonies is - Expressed in terms of per lung. Experiment I numbers (rrz) are shown in parentheses.

Figure 2 shows the influence of methyl β-D-rays on the number and size of lung colonies of BL6 cells. It is a graph showing the influence of pre-administration of kutosito. Methyl β-D-lactocyto (administration) A dose of 1 ml) was injected intraperitoneally into C57BI mice. 10 minutes later, BL6 Melano The tumor cells were injected intravenously.

Lung colonies were counted and sorted on day 19. Group A is control animals (methyl β-D-lactocyto); groups B and C represent methyl β-D- Represents animals injected with 0.25M and 0.5M Lactonde, respectively. each glue For each group, column 1 represents the total number of colonies and column 2 represents the number of colonies >1 mm in diameter. and column 3 represents the number of colonies with diameter <1 mm. The number of experiments is expressed as rnJ. It is.

Figure 3 shows the expression of tumor-associated carbohydrate antigen (TACA) defined in tumors. It is a graph showing the survival rate of cancer patients with or without cancer. Panel 3A is , in lung squamous cell carcinoma measured by monoclonal antibody MIA-15-5. Expression of H/Le'/Leb antigen. Panel 3B shows the results using antibody FH6. Figure 2 depicts sialosyl-Le' expression in intestinal cancer. Panel 3C uses antibody TKH2. Figure 2 represents sialosyl-Tn expression in colon cancer. Panel 3D shows ovarian cancer patients. It represents the concentration of Sialonru Tn in serum.

Figure 4 shows that melanoma cell adhesion to LacCer is This is a graph showing that this is due to Ce r interaction.

The metastatic order is BL6>FI O>Fl>>Wa4. Panel 4A is L The order of melanoma cell adhesion to acCer-coated solid phase is shown. Panel 4B is Melanoma cell attachment to LacCer/fibronectin (FN) co-coated solid phase Indicates the order of arrival. Panel 4C shows integrin-dependent adhesion.

Figure 5 shows LacCer (panel 5A) and endothelial cells (HuVEC) (panel 5A). 5B) melanoma cell (BL6) adhesion to La, cCer and G It is a graph showing that it is suppressed by M3.

FIG. 6 is a graph showing the metastasis suppressing effect of methyl (Me)-β-lactocyte. . After intravenous injection of tumor cells, PBS (A); 0°25MMe-β-lacto Cyto (B); 0.5M Me-β-lactose (C); 015M Lactose ( D); 0.25M N-acetyl raftosamine, (E); or 0.5M Me -β-galactoside (F) was injected intraperitoneally.

FIG. 7 is a graph showing H-Le' and H-H interactions.

Panel 7A shows Hl-liposome binding to various glycolipids.

Panel 7B shows Le'-liposome binding to various glycolipids.

Figures 8A-8D show inactive (panels 8A and 8C) platelets and anti-MP- Flow of activated (panels 8B and 8D) platelets using 140 monoclonal antibody Cytophotometric profile.

Figure 9 shows the binding index of platelets to fluorescent beads coated with various GSLs. It is a graph. Hatched bars represent non-activated platelets; oven bars represents activated platelets.

FIG. 1 is a graph showing the influence of various monoclonal antibodies. The horizontal axis represents the suppression rate . Column 1 represents anti-GMP-140-mAb, 10P62; column 2 represents anti-GMP-140-mAb, 10P62; Represents the monoclonal antibody CAl9-9: Column 3 is anti- Noarosyl-Le' monoclonal antibody, representing 5NH4: and column 4 represents normal mouse IgG.

Figures 11A to 11D show experimental systems demonstrating dynamic adhesion of cells in a flow system. Showing the stem. Panel IIA shows the structure of the laminar flow chamber. Panel l 1. B shows a cross section of the laminar flow chamber. In this laminar flow chamber, the flow chamber - The main body (16) has a coverslip (on which cells or adhesion molecules (9) are immobilized). 3) is firmly fixed. Panel IIC is the assembly of the recording system. - Show the whole thing. Panel IID is a tumor cell suspension passing over a cell layer or adhesion molecules. It is a schematic diagram showing the flow of liquid.

Figure 12 shows interleukin-1-activated human septum endothelial cells in a dynamic flow system. Effect of various monoclonals on the adhesion of human colon cancer Co1o205 cells to cells. It is a graph showing the influence of antibodies. White circles are a mixture of unrelated mouse IgG and IgM. The black triangle represents the data for the mono-alonol-le”i. Data for clonal antibody CA19-9 are shown, and the white squares represent sialosyl-Le'. represents the data of monoclonal antibody 5NH4 against the monoclonal antibody 5NH4. Data of monoclonal antibody FH7 against Le'll and dine allosyl Le' The black angle of view is that of unrelated mouse IgG+IgM and non-activated endothelial cells. Data for mixtures are shown.

Figure 13 shows the binding of mAb to HL60 cells and the effect of sialidase on it. Show the impact of Binding activity was determined by flow cytometry. Horizontal axis dilog fluorescence Strength. Vertical axis/relative cell number. Panel C: Solid line is stained with mAb SNH4 as the primary antibody. The results are for colored cells, and the dotted line is mouse I'gG+I as the primary antibody. These are the results of control cells stained with gM (10 μg/ml). Panel B, - next anti- mAb SNH3; control as in panel 8. Panel C: Solid line is Newcastle Cells stained with mAbsNH4 after treatment with NDV sialidase. The dotted line represents control cells (panel A except after sialidase treatment). Similar results). Panel D: mAb SNH3 after NDV sialidase treatment The control is the same as panel C. Panel E: Vibrio cholerae (V C) Staining with mAb SNH4 after sialidase treatment. Panel F: VC-alidase treatment Stained with mAb SNH3 after surgery. (Confirmed by both SN H3 and 5NH4) ) Expression of SLe' was completely abolished by both NDV and VC sialidase. Ta.

Figure 14 shows HL60 cells on E-selectin coated plates in a static system. Indicates adhesion. The horizontal axis indicates the type of processing. The vertical axis is the fine line relative to untreated control cells. The cell adhesion rate is shown. Panel A. Effects of two different sialidases. Panel B, anti-Le’ and anti-3Le’mAb alone and in combination (90 min incubation at 37°C). vation).

Panel C: Effect of NDV anase + mAb. Panel A: (at the terminal Gal NDV sialidase (which cleaves α2→3 aronol) eliminates the SLe’ structure. to eliminate reactivity with mAb SNH3 and 5NH4 (see Figure 13). , the adhesion did not disappear. vC and Arthrobacter ureafanoens (A) V) Sialidase abolished adhesion. Panel B: Anti-5Le’ mAb is anti- The effect was smaller than that of Le' mAb. The combination of both types of mAbs It was the most effective. Panel C adhesion is NDV annoase + anti-L e’ It was most effectively inhibited by mA b.

Figure 15 shows HL60 for E-selectin coated plates in a dynamic flow system. Showing cell adhesion. Truncated E-selectin was placed on a plastic plate. Apply to the marked area (approximately 0.5 cm in diameter) and bond under known wall shear stress. were assayed using the methods described herein. The horizontal axis is the shear stress (dynes/cm' ). The vertical axis is the number of cells that adhered in 3 minutes. Panel C: Solid line is control (not treatment) cells; black triangles are cells treated with NDV-alidase; black circles are cells treated with VC sialidase. -ase and Shiratama-kaku are AU sialidase. Panel B: White circles are control, black triangles are anti-control. -8Le’ IgCz mAb SNH4-containing cells; black circles indicate Anti-Le' IgMmAb FH2; and the white ball corner is anti-Le' IgGx mA bsHI. Panel C1 white circles are control; black triangles are NDV aridase; black circles are mAbsHl, and white ball corner are NDV annoase + mAbsH1. panel D. White circles are control, black circles are (1:I) mixture of mAb SNH4 and FH2, and Shiratamajiku is a (1:1) mixture of mAb SNH4 and SHI. NDV nah Cleavage of α2→3-aronolation at the terminal Gal by lidase causes some adhesion. Although reduced, adhesion remained at low shear stress. On the other hand, VC and AU serial Dase strongly suppresses adhesion (Panel 15A), internal cyanide (NDV) (not affected by lidase) was shown to be important. This means ( i) NDV anase + mAbsHI strongly inhibited adhesion and (11 ) Anti-8Le’mAb SNHJ + anti-Le’mAb FH2 or SHI is 5NH4 This is evident from the fact that it suppressed adhesion more strongly than alone (panels 15B and 15D). It is.

Figure 16 shows Co1o201 with and without sialidase treatment. The reactivity of cells with various mAbs is shown. Co1o201 cells are anti-5Le' ImAb CAl9-9 and NKHI (Panel A), anti-Le' mAb CA3F 4 (Panel B) and reaction with anti-3Le'I ImAb FH7 (Panel C) It was highly sexual. Reactivity with CA19-9 was determined by NDV aridase (panel D). and was abolished by VC sialidase (Panel G). Reaction with Ca3P2 The response was slightly increased by NDV and VC alidase (Panels E and H) . Reactivity with FH7 was not changed by NDV aridase (panel F); and was slightly reduced by VC aridase (Panel I).

Figure 17 shows Co1o20 for E-selectin coated plates in static system. 1 adhesion is shown. The horizontal and vertical axes are the same as in FIG. 14. Panel A Two types of shears Effect of lidase (90 min incubation at 37°C). Panel B: Nari Effect of Dase (incubation for 18 hours at 37°C). The cells are first 0.5 % formaldehyde for 10 minutes at room temperature. Panel C; Sialidase treatment Effect of post-treatment with mAb. N cleaves α2→3 sialon with terminal Gal DV-alidase had no effect on adhesion, whereas VC and AU sialidases, which cleave alkyl acids, abolished adhesion (panel B).

In panel C, for VC or AU-inoadase + mAb CA3F4, the Effective inhibition was also observed.

Figure 18 shows Co1o for E-selectin coated plate in dynamic flow system. 201 cell adhesion is shown. Adhesion assays were performed as described herein.

The horizontal and vertical axes are the same as in FIG. 15.

Panel A: White circles are control, black circles are NDV sialidase; white circles are AU noalidase , and the black triangle is VC sialidase. Panel B White circles are control; black circles are anti-8L. e’　ImAbCAl　9-9　; White ball corner is anti-5Le’　ImAbFH7 ; and the black triangle is anti-Le' mAb CA3F4. Panel C: white circles are controls; The black circle is Ca3P2. Black triangles are VC-alidase, white triangles are VC-sialidase+ CAl9-9. Then, CC equilateral triangular VC nonaguse-←CA3F4 (adhesion is The strongest combination of this combination was S), which was suppressed by J). Panel D: white circles are contrast: black Triangle is N I) V Sialidase 1 Black inverted triangle is CA3F4: White road triangle ijl N D V Noalidase CA19-9. Black circles indicate NDV sialidase 1FH7+ The white triangle indicates NDV sialidase + CA3F4 (adhesion is the best with this combination. was also strongly suppressed).

Figure 19 shows the ELAM application plate in a dynamic flow system under various shear strength conditions. Newcastle disease virus (NDV) sialida for L60 binding to Vibrio cholerae (VC) sialidase or ■ηAbSNH4 or S) (Indicates + effect. Vertical axis represents cell binding to untreated control cells. 5 μg/ml and NDV sialidase at 0.2 U/ml. Alidase was used at O.IU/ml. Each point is the average of three experiments.

Shear stress 15.5.7.75.3. I3.1.56 and 0.78 dynes/cm2 The number of untreated cells combined in 4.5.27.109.6.206.2 and and 283.8 pieces/m2.

Figure 20 shows various branched sugars. )＼Iblint sugar, Le’ / SLe’, is shown as structure l. Glycolipids containing such structures are used to treat colon cancer. human erythrocyte-derived construct 5 by separation from (Watanabe et al., J.

13io1. Cbem, 254: 8223.1979) prepared from nglioside. Construct 2 is the αI-3 fragment of compound 6 derived from human placenta. Obtained by conversion. However, construct 2 It did not show high affinity binding. Structures 3 and 4 have Le' and Noary in the same molecule. Analogue with high affinity binding site (construct structure 3) or the hybrid molecule Le'/SLe'', which is a positional isomer of structure l. .

Figure 21 shows various types of glycoliposomes coated on the plastic surface. The adhesiveness of N S-1 cells expressing E-selectin is shown. Panel 21A is , the above relative contact in a dynamic flow system under medium shear stress conditions (7,75 dynes/Cm') This is the result of wearing. The first seven bars are for each glycoliposome displayed. The relative adhesion of NS-1 cells to SLe' is shown. cpa I has the structure shown in Figure 20. There is a structure 1, and CpdlI is structure 2 in FIG. Rods 8 to 10 are L A mixture of different compounds labeled e' is shown. The value of relative adhesion is SLe'- It is expressed as a comparison with the case where the adhesion of liposome is set as 100%. Values are from 5 measurements. is a constant average. Panel 21B was tested under high shear J conditions (11,8 dynes/am' ) shows the relative adhesion of the same NS-1 cells. This value is It is expressed in terms of adhesion with respect to Values are the average of 5 measurements.

Figure 22 shows various glues applied to plastic plates at different shear stress conditions. Relative adhesion of NS-,1 cells expressing E-selectin to licoliposomes Show your gender. CPDI and CPDII are structures I and 2 in FIG. C.P. Increased adhesion for DI-coated plates was only seen at moderate to high shear stress conditions. Ta. The vertical axis is the relative adhesiveness to SLe' liposomes. The horizontal axis is the dynamic flow. is the wall shear stress (dynes/cm') at DSI is Ginoarosiru ■ Represents an antigen.

Figure 23 shows various glycolipids applied to plastic surfaces at different glycolipid concentrations. The number of cells attached to the soma (per mm'). Structure l in Figure 20 is a high shear Due to the stress, much more E-selectin-expressing cells were able to attach than on the SLe” coated plate. wear it. This difference is not clear at low shear stresses. The vertical axis is the number of connected cells/m 2, and the horizontal axis is the glycolipid concentration (unit, μM). Each point is the average of 5 measurements. It is average.

Figure 24. Glycoliposomes with a mixture of SLe' and various other glycolipids. Figure 3 shows the adhesion of NS-1 cells expressing E-selectin to. The vertical axis is l-region light This is the number of adherent cells. The black circle is SLe'+SPG. White circles are SLe ’　+82. The black triangle is SLe' 10Le'. The white triangle is SLe’ There is +Le’t’. Each point is the average value of 5 measurements.

Detailed description of the invention As described above, according to one aspect of the present invention, a method for suppressing tumor cell metastasis and invasion is provided. Methods and compositions are provided. Various tumor cells can metastasize, which means that they can spread to two distant sites. Has the ability to form secondary tumor colonies. Melanoma is the source of malignant tumor cells. , lung cancer, breast cancer, colorectal cancer, and genitourinary cancers such as bladder cancer and prostate cancer. . In the present invention, the metastatic potential of a tumor cell (i.e., the ability of a tumor cell to metastasize) is defined as ( a) Tumor-associated carbohydrate antigen [TACA (herein, TACA also refers to LACA) (b) Antibodies against these TACAs: (C) This Oligosaccharide component of TACA; (d) Linluridine or TACA-supported glycos TACA such as multivalent complexes of fingolipid (GSL) liposomes or similar +U combination of oligosaccharide component of eel TACA or (e) using a TACA mimic This is suppressed by In general, unless otherwise specified, tumor cells and white blood cells are Because both bind to endothelial cells through carbohydrate structures, they are virtually equivalent. It is.

TACA epitopes bind tumor cells through interactions with endothelial cells, platelets, and basement membranes. Tumor metastasis and invasion are thought to occur by playing a major role in cell adhesion. . The adhesion mechanisms include carbohydrate (CHO) CHCI-CHO interaction, CHO-lectin This is thought to be due to CHO-selectin interaction or CHO-selectin family interaction.

Adhesion of various tumor cells to non-activated endothelial cells initially occurs in a carbohydrate-carbohydrate phase. interaction, which triggers activation of endothelial cells and ELAM-] and Expresses selectins such as GMP-140 (Kojima and Hakomo ri, Biol, Chem, 266:17552, +99]; Kojima et al. , J. Biol.

Chem, 267:17264.1992; Hakomori.

Histochem, J., 24 Near 71, 1992). Subsequently, the activation LECCAM or serum is mainly used for the adhesion of various tumor cells to endothelial cells and platelets. mediated by the lectin superfamily (e.g. ELAM-1 and GMP-140). Exists.

Tumor cell adhesion mediated by sialosyl-Le' suppressed by noclonal antibodies (FH6, C5LEXSSNH3 and 5NH4). Therefore, tumor cell adhesion mediated by monosialosyl-Le''I is linked to its epitope. Monoclonal antibodies against (CAl!J-9, C3LEASNKHI and NK H2) is suppressed by (Handa et al., Biochem, Biophys, Res, Commun, l　8　]:1223.1991: Kojima et al. Biochm, Biophys, Res, Commun, +82:1288 ．． 1992+Hakomori, Histochem, J., 24.771. (see 1992).

For endothelial cells, mainly l-channel sialosyl-Le'' and (to a lesser extent) Adhesion of Co1o205 tumor cells expressing Aronleu Le' -Le' monoclonal antibodies and (to a lesser extent) anti-sialosyl-Le' Inhibited by monoclonal antibodies. These findings are from Siaron Le' In addition, sialosyl-Le’ is also used for ELAM-1 and GMP-140 (previously C Referred to as D62 or PADGEM, as E-selectin and P-selectin This suggests that it is an important ligand recognized by .

Adhesion of tumor cells to activated endothelial cells is determined by monosialosyl Le'll and di It is currently known that it also depends on the recognition of monocyaronrue. Both Le'll and Zin-Allosyl-Le' stimulate selectin-mediated activation of the endothelium. Connection of various epithelial cancer cells (particularly colorectal, gastrointestinal and pancreatic) to cells or platelets. This was confirmed using the monoclonal antibody FH7, which is known to strongly inhibit It will be done.

In particular, GMP-140 is effective in α-granules of platelets or Weibel of endothelial cells (EC). -Major selectin located in Weibel-Pallade body ( LECCAM). When these cells are activated, GMP-1,40 It rapidly redistributes to the surface where GMP-140 is expressed on blood cells or tumor cells. plays an important role in the adhesion of platelets or ECs to certain carbohydrate epitopes As a result, the aggregation of platelets or tumor cells or their contact with the capillary endothelium The clothes will grow. According to the inventors, GMP-140-mediated cell adhesion It appears to be involved in the initiation of metastatic attachment and the initiation of inflammatory processes.

In addition, ELAM-1 is interleukin-1, TGF-β, TNF-α or Expressed on endothelial cells after activation with popolysaccharide. ELAM-1-mediated cell adhesion also It appears to be involved in the initiation of metastatic attachment of tumor cells.

Therefore, according to another aspect of the invention, GMP-1, inter alia at the tumor cell site 40-mediated or ELAM-1-mediated inhibition of cell aggregation or adhesion. In the present invention, GMP-140-mediated or ELAM-1-mediated cell aggregation or adhesion is induced by (a) Le“/ Hybrid sugars such as S L e'; (b) hives such as Le' and SLe' A mixture of saccharides that is a component of lid sugar (a); (C) monosialosyl=Le” i Le', Lew, Mono/Aro/Lou Le'II, Shiso Aron Le'Also is sialosyl Le'; (d) Le' / SLe' etc. ), Mono/Allo/Lu Le’ I, Le”, Le’, Mono/Allo True Le 'IT, an antibody that specifically binds to Shisoalone Le'' or Sialosyl Le°; (e) Antibodies against components of hybrid sugars, such as in particular against SLe' and Le' (f) Hybrid sugars such as Le'/SLe', mono/allots Le’i Le’, Le’, Mono/Aro/Lou Le’ll, Shiso Aron Oligosaccharide component of Le’ Lc’ or Noaron Le’: (g) Le’/SL e’ etc./＼Iburi, sugar, mono/Aron Rou Le’ l Le’, Le’ , Monosialon Le'lr, Shisoalone Le' or Sialosil Le' Complexes or complexes of oligosaccharide components, and (h) hybrids such as Le'/SLe' Red sugar, Mono/Allo True Le' I, Le', Le', Monosialo/L e'll, disialosyl Le' or sialosyl Le' mimetics. This can be suppressed by

In the present invention, tumor metastasis and invasion are inhibited by blocking tumor cell adhesion. Inhibited by reducing or eliminating the spread of metastatic cells.

Furthermore, in the present invention, tumor metastasis and invasion are caused by (1) (a) contact of tumor cells with platelets; (b) adhesion of tumor cells to tumor cells via platelets; (C) adhesion of tumor cells to tumor cells via platelets; Adhesion of tumor cells to ECs and (d) ECs to tumor cells via GMP-140 GMP-140-mediated tumor cell aggregation or adhesion at the tumor site by direct adhesion of; (2) direct adhesion of cells to ECs via ELAM-1 at tumor cell sites; ELAM-1-mediated tumor cell aggregation or adhesion can be suppressed to a minimum. Ru.

Furthermore, in the present invention, inflammation includes (a) adhesion of leukocytes to platelets, (b) Adhesion of leukocytes to endothelial cells (EC) via the plate, (C) EC via selectins (d) Possibility of inflammation due to direct adhesion of leukocytes to and (d) migration of leukocytes through endothelial cells. Inhibiting GMP-140-mediated leukocyte aggregation, adhesion, or migration at a certain site more minimized.

TACAs suitable for use in the present invention are those that exhibit differential prognostic significance (i.e. , TACA), which has a clear correlation with invasiveness or metastasis. In the present invention, The above-mentioned TACA may be related to the invasiveness of similar tumors showing expression/non-expression of such TACA. , can be identified by comparing metastatic potential and clinical prognosis. For use in the present invention Preferred TACAs include H/Le'/Le''', Noarosilu Le' (SA -Le' or SLe'), Le', Le', monosialosyl-Le' 1 (SL e’ or S A-L e’) and Siaron Lu Tn (SA -Tn or 5Tn). Such TACA derivatives include Le'/ SLe', Le' dimer, non-allotrue Le' dimer, trifucotle■, e ', non-allo-true LeI and mono-alonol-Le'II, and other hybrid sugars Includes types.

As mentioned above, TACA used in the present invention shows differential prognostic significance. example For example, the differential prognostic significance (confirmed by monoclonal antibody MIA-15-5) Tumors that express H/Le'/Le' antigens do not express such antigens. It is clear that the prognosis of a midtone tumor is much worse than that of a tumor. for example, As shown in Figure 3A, patients with squamous cell lung cancer expressing H/Le'/Le' The 5-year survival rate was 11% (i.e., mortality rate 89%); Patients who do not express comparable H/Le'/Leb have a survival rate of It was about 62%.

Regarding tumors showing expression or non-expression of Noalonoru Le' and Sialonoru Tn antigens However, similar results were obtained. More specifically, as shown in Figure 3B, sialo Patients with colon cancer expressing Sil-Le' have a 5-year survival rate of only 15%. In contrast, patients who did not express the above-mentioned antigen responsible for this The survival rate was approximately 50%. In a separate study, expression of noalosyl-Tn The 5-year survival rate for patients with early-stage colon cancer was 100%; The survival rate of patients expressing Noalosyl-Tn was 75% (see Figure 3C). . A patient with ovarian cancer expressing sialosol-Tn antigen, as shown in Figure 3D. A similar but clearer difference was observed in patients who did not develop the disease.

In addition, as described above, an appropriate antibody or mixture of antibodies against TACA can be used with the present invention. Can be used in bright light. The antibodies used here include monoclonal and polyclonal antibodies. Null antibodies include both intact molecules, fragments of such molecules or their functional equivalents. It can be a thing.

Antibodies can be constructed genetically. As an antibody fragment, for example, F(ab')r , Fab', Fab and Fv.

Simply put, polyclonal antibodies are produced by collecting serum from an animal after immunizing it. It can be obtained by Immunization can be carried out, for example, in rabbits, rats or mice. It can be administered systemically, such as by subcutaneous, intravisceral or intramuscular injection. General Generally, it is preferable to perform one or more booster immunizations after the initial immunization and before serum collection.

Such methodologies are well known and described in numerous publications.

Although polyclonal antibodies can be used in the present invention, monoclonal antibodies are preferred. Delicious. Monoclonal antibodies suitable for use in the present invention include murine or Human-derived polyclonal antibodies or a combination of human and mouse antibody parts chimera such as a mouse antibody (antigen-binding region of a mouse antibody + constant region of a human antibody) Contains antibodies. Human and chimeric antibodies are produced using methods known to those skilled in the art. can. Human antibodies and chimeric human-mouse antibodies are more easily administered clinically than mouse antibodies. This is advantageous because it is less likely to cause the production of anti-antibodies.

Monoclonal antibodies are generally described by Koehler and MiIstejn. Method (Nature256:495.1975; EurJ, Immunol, 6 :511.1976) as well as the first of Koehler and Milstein. Various techniques that improve upon the method of Harlow and Lane (eds.), “Ant ibodies": A Laboratory Manua Bi, Col1d See pring Harbor Laboratory+ 1988; this document (the entire disclosure of which is hereby incorporated by reference) .

Briefly, the lymph nodes and lymph nodes of animals immunized with one type of TACA or oligosaccharide components. and/or spleen with myeloma cells to create a hybrid cell line (hybridoma J or “clone”). Each hybridoma contains a single species of immunoglobulin It is possible that the cells secrete secretion and undergo endless cell division, like myeloma cells. this It may be desirable to conjugate such molecules to carriers to increase immunogenicity. . Transfer to an appropriate ff carrier (J, keyhole rimbet hemonoanin, zyroglobulin) , Uno serum albumin and their derivatives.

An alternative to producing monoclonal antibodies via hybridomas is Lyophage and bacteria (e.g. 5asLry etc., Proc, Nat 1.A cad, Sc, USA 86:5728.1989; Huse et al., 5ci ence246:1275.1.289) for monoclonal antibody expression. The second is to create a brary. Selection of antibodies exhibiting appropriate specificity is within the skill of the art. This can be done in a variety of obvious ways.

Representative examples of monoclonal antibodies suitable for use in the present invention include MIA -15-5 (M i V a k e and Hakomor i, Bioche m, 30:3328, +991), Hakomori, Advances I n　Cancer　Re5earch52:257-331.Quoted in 1989 Examples include monoclonal antibodies.

As noted above, any suitable oligosaccharide component of TACA can also be used in the present invention. Book The term "oligosaccharide" as used in the specification includes natural oligosaccharides, synthetic oligosaccharides, A saccharide and any mimetic derivative thereof comprising a portion of the TACA oligosaccharide component included.

Additional oligosaccharide components useful in the invention include lactose and methyl β-p-la Kutonodo, Lacto-N-tetrose (Galβl-3GIcNAcβ] → 3Ga 1β] → 4GIc) and lactose derivatives such as phenyl β-thiolactonodo or included. For example, both components suppressed melanoma cell metastasis in mouse lungs. It turned out that I would. Ethyl or phenyl lactone and methyl or ethylthio Lactoose derivatives including lactones can also be used. The above lactose derivative melanoma cell GM3 ganglionode interaction with lactonorceramide in EC It is thought to block the attachment of melanoma cells to EC by inhibiting It will be done.

Other oligosaccharide moieties that are suitable for inhibiting the metastatic potential of certain tumor cells may By determining the structure of the specific carbohydrate chain(s) involved in transfer ability. Can be identified. Identification of carbohydrate-containing molecules involved in tumor metastatic potential and various methods including by the use of inhibitors of glycosyltransferases. It can be done with

Structures of carbohydrates bound to lipids or proteins can be directly probed by electron impact and decomposition. (E+) and mass spectrometry including fast atom bombardment (FAB) and methylation analysis (e.g. Nudelman et al., J. Bi.

1, Chem, 261:5487.1986). Can be determined. Degradation analysis can be performed chemically and/or enzymatically, e.g. You can do more. The carbohydrate sequence suggested by degradation analysis is After analysis (Hakomori, J, Biochem, 55: 205.1964) , chemical ionization mass spectrometry of permethylated sugars (Stellner et al., Arch, B iochem, Biophys, 155:464.1974; Leverye determined by tal, Mcth, Enzymol, 138+13.1987) Can be determined.

In place of or in addition to the above techniques, El mass spectrometry can be used to analyze permethylated glucans. It can be carried out after proper degradation of intact glycans (Kan et al. nagi et al., J., Bjol, Chem.

259:8444.1984: Nudelman et al., J. Biol. , Chem, 263:13942.1.988). Carbohydrate sequence uniformity is Proton NMR spectroscopy and FAB mass spectroscopy of scars or methylated glycans can be indicated on the basis of various chemical and physical criteria, including: once carbohydrates Once the sequence has been determined, one skilled in the art will be able to determine the appropriate origin for inhibiting the metastatic potential of the tumor cells. The obvious choice would be Gosu.

As briefly mentioned above, lysyl lysine or TACA-bearing glycosphingoli Suitable polyvalent complexes with GSL (GSL) liposomes (or glycoliposomes), etc. Complexes of TACA or their oligosaccharide components can also be used in the present invention.

The components of the conjugate can be covalently linked to each other directly or through a linker group. between the ingredients Direct reactions are possible when each has substituents that can react with the other. for example, -Nuclear groups such as amino or sulfhydryl groups on components are Leaving groups that can react with carbonyl-containing groups such as anhydrides or acyl halides; For example, it can react with alkyl groups containing halides.

It may be desirable to covalently bond the components through a linker group. linker group serves to increase the coupling efficiency by increasing the chemical reactivity of the substituents. Tasu. Increased chemical reactivity also makes it difficult to use functional groups in unavailable components. becomes easier. For example, carboxyl groups can be activated. carboxyl group activity includes the formation of "active esters" such as succinimine esters. .

The term "Activated ester J" refers to an ester that is highly reactive in the claimed nuclear substituent reaction. It is known that it means a group of people.

It may also be desirable to produce complexes in which the components are non-covalently bound. Ru. For example, one or more TACAs can be combined with glycosphingolipid (GSL) liposomes. can be incorporated into the external surface of the system.

It may be desirable to increase the in vivo half-life of oligosaccharides.

As disclosed in the present invention, the oligosaccharide can be converted into a straight-chain polysaccharide such as polyethylene glycol. Can be attached (ie, co-attached) to amphoteric polymers. Oligosaccharide-polyethylene glyco A typical example of a method for producing a scunonimine complex is Reacting oligosaccharides with polyethylene glycol having terminal amino groups That is. The latter compound has the general formula N)4. -(CH, -CH2-0)n-CH .

(where n typically averages from 44.7 (i.e., molecular weight about 2,000) to 112 ．． 9 (ie, molecular weight approximately 5,000).

Furthermore, selectins, ELAM-1 or GMP 140. cell adhesion mediated by is a sialyl reaction caused by the lectin sequence region present in the N-terminal region of the selectin molecule. It is based on the recognition of type 1 and type 2 chains of the lactose and fuconolated lacto series. Lectin domain blocking activity may be more effective than natural epitopes. Either structure is effective for the present invention. Similar to the surface structure of natural epitopes Such non-naturally synthesized compounds show better blocking activity than carbohydrate-dependent adhesion. products, such as Sialon Le' or Sialosyl Le'l or the same "mimetic" Such non-naturally synthesized compounds, which are referred to as "products", are considered.

Useful mimetics include, for example, trifluoro-thi-fucose, N,-1-rifucose, fluoro-acetyl-glucosamine or natural sialon-ru 1, e', mononoalone Noalic acid analogs at the same distance and spatial location as found in Le' I and 11 and heterocyclic or aromatic ring structures with fucose analogs, or trifluoro- L-fucose, N-trifluoro-acetyl-glucosamine or N-1-rif H containing fluoro-acetyl-neuraminic acid/with allo-true Tn analogs Sialosyl-Le' or monosialon-Le having /Le'/Lel' structure '　I or II, but not limited to these.

Therefore, cell contact via tumor-associated carbohydrates is more efficient than natural epitopes. Modified carbohydrate epitopes or surface structures of carbohydrate epitopes that block Other "mimics" that are similar are within the scope of the invention.

Metastatic potential of tumor cells and GMP-140-mediated or ELAM-1-mediated cell aggregation or Inhibition of adhesion can be achieved both in vitro and in vivo, e.g. by treatment of isolated tumor cells or tumor bearing. To treat disease processes involving host hosts and GMP-140 or ELAN1-1. Used in a wide variety of ways.

As mentioned above, with respect to in vitro, the present invention provides tumor cell metastatic potential within biological samples. A method for suppressing is provided. This method analyzes biological samples that have (a) different prognostic significance; (b) an antibody that specifically binds to the antigen; (C ) an oligosaccharide component of the antigen, (d) a complex of the antigen or oligosaccharide component, and (e ) suppressing the metastatic potential of a sample selected from the group consisting of mimics of tumor-associated carbohydrate antigens; the step of incubating with at least one agent that

Furthermore, with respect to in vitro studies, the present invention provides GMP-140-mediated Alternatively, methods of inhibiting ELAM-1-mediated cell aggregation or adhesion are also provided. Head The method involves converting a biological sample into (a) a hybrid sugar such as Le'/SL e''; (b) L Sugar component of hybrid sugar (a) such as e’ and SLe”: (C) Monosialosyl -Le' l Leo, Le', monosialosyl - Leo II, monosialosyl -Le' or Sialosil ■, e': (d) Noive such as Le' / SLe' Lid sugar or its component sugars, mononoalone Le'I, Le', Le', Monosialosyl-Le'' II, ZinAron Le' or Sialosyl Le (e) Hybrid sugars such as Le''/SLe'; Monosialocyl-Le'll , an oligosaccharide component of shisoallosyl-Le' or sialosyl Le'; (f) Le'' /1 hybrid sugar such as /SLe', monosialosyl-Le'I, Le', Le”, monosialosyl-Le’ll, shisoalosyl-Le” or sialosyl a complex of SLe' or a complex of oligosaccharide components: and (g) SLe'/Le' Hybrid sugars such as monosialosyl-Le' 1. Le’, Le f, mono Pseudo of sialosyl-Le’ll, dine-allosyl-Le’ or sialosyl-Le’ at least one drug that inhibits cell aggregation or adhesion selected from the group consisting of and incubating with the agent.

Suitable biological samples include microorganisms in biological fluids such as blood, urine, lymph, synovial fluid, and spinal fluid. These include cell cultures and cell suspensions. TACA, oligosaccharide or complex thereof is one Generally the final concentration is about 0. 1 to IM, typically about 0. Incubate at 2-0.5M will be applied. Incubation is typically 5-15 minutes at 37°C. cormorant. After processing the biological sample, this sample can be injected or implanted into an animal, e.g. Confirm the sexual suppression effect.

Further, according to the present invention, there is provided a method for suppressing tumor cell metastasis in warm-blooded animals such as humans. law is provided. The method provides a method for testing warm-blooded animals with (a) tumor-associated tumors that exhibit different prognostic significance; (b) an antibody that specifically binds to the antigen; (c) an antigen of the antigen; (b) an antibody that specifically binds to the antigen; oligosaccharide component, (d) complex of the above antigen or oligosaccharide component, and (e) monosialon Lou Le “■, Le’, Le’, Monosiaron Lou Le’ll, Shisoarosi of a sample selected from the group consisting of Le-Le' or sialosyl Le' mimetics. administering an effective amount of at least one drug to inhibit metastasis. Ru.

Similarly, the present invention provides for GMP-140-mediated or Also provided are methods of inhibiting ELAM-1 mediated cell aggregation or adhesion. This method is , to warm-blooded animals, (a) hybrid sugars such as Le''/SLe': (b) Le' and component saccharides of (a) monosialosaccharide such as SLe'', (C) monosialosyl -Le” I, Le”, Le’, Monocyaron Ru Le” II, Gin Alloshi Hybrids such as Le” or Sialosil Le’; (d) Le”/SLe’, etc. sugar, monosialosyl-Le' L Le', Le'', monosialosyl- Le" II, specifically binds to di-allosyl-Le' or sialosyl Le' Antibody; (e) Hybrid sugar of Le'/SLe' sugar, monosiaron-Le ″11 Monocyalone Le’Il5Le’, Leo, Shisoalosyl- Oligosaccharide component of Le' or sialosyl Le'; (f) Le' / SLe' etc. Noybrid sugar, monosialosyl-Le'L Le', Le', monosialosyl-Le'L Composite of allosyl-Le'll, di-allosyl-Le' or sialosyl Le' complexes of bodies or oligosaccharide components, and (g) Le'/SLe'' etc. Liver sodonodomonosialosyl Le' l5Le', Le', monosialosyl - A group consisting of mimics of Le'll, Jinaron Le' or Sialosyl Le' A small amount effective to reduce metastatic potential at the tumor site in warm-blooded animals selected from comprising administering at least one drug.

Also, according to the present invention, GMP-140-mediated cells at sites of inflammation in mixed-race animals A method of inhibiting aggregation or adhesion is provided.

This method provides a warm-blooded animal with: (a) Nolbu-J nodosaccharides such as Le'/SLe'; (b) Component sugars of /% Ibri nodosaccharide (a) such as Le'' and SLe'; (C ) Monosialosyl-Le”■, Leo, Le”, Monosialosyl-Le’II , Jino Aron Lou Le'Mata (Masiarosil L e' + (d) Le'/SL Hybrid sugars such as e', monosialosyl-Le' I, Le', Le', monosialosyl-Le’ll, di-allosyl-Le” or sialosyl-Le’ Antibodies that specifically bind.

(e) 71-ib 1 lusodosugar, monosialosyl-Le' such as Le' / SLe° l Le', Le', monosialosyl-Le'll, shisoalosyl-Le' Or oligosaccharide component of sialosyl L e' + (f) Sl-e'/Le' etc. hybrid sugar, monosialosyl-C'I, Le', Le', monosialo Sil-Le'11, Nsialon-1-e' or a combination of Sialosil Le' Consolidation or complexes of oligosaccharide components, and (g) novel compounds such as Le' / SLe' 1 lunodosugar, monosialosyl-Le'i Le', Le', monoalosyl Lu Le’l+, Jinaro True Le’ or Sialosil Le’ mimic physical power 1 etc. Effect of reducing inflammatory properties at inflammatory sites in warm-blooded animals selected from the group t4 '] f,; administering an amount of at least one drug.

In both methods, TACA oligosaccharides or complexes thereof are generally added at about 0.1 to The concentration of IM, typically about 0. Administer at a concentration of 2-0.5M. For example, In vitro and in vivo studies in human animals (based on which optimal dosages for specific substances can be determined) It will be clear to those skilled in the art how to determine the be able to. Typical V) includes, for example, the pleural or peritoneal cavity, the bed of the resected tumor; Also, administer intravenously or intracorporeally to the site of inflammation.

The above-mentioned TACA, antibodies, oligosaccharides or derivatives are Administer in combination with a carrier or diluent such as physiological saline.

Additionally, agents that inhibit or reduce metastasis may be combined with immunotherapy or chemotherapeutic agents. Drugs that reduce inflammatory properties should be administered in combination with anti-inflammatory substances. be able to.

When such drug and substance combinations are desired, each compound in turn is They can be administered simultaneously or in combination and as a single composition. Before and after administration Diagnosis such as CAT scan is performed after administration to confirm the effect of suppressing metastatic or inflammatory disease. can.

Measurement of adhesion or aggregation of tumor cells to other cells (e.g. ECs) or adhesion or One in vitro system to confirm successful suppression of aggregation is M, B, La described by Wrence et al. (BIood70: 1284, I 987). , and as shown in Figures 11A, 11B1, 11C, and 11D. A similar dynamic flow system.

A parallel plate laminar flow chamber (1) (shown upside down for convenience) is inserted through a tube (18). is used in conjunction with a pressure pump (2) to reduce the flow shear stress present in the microvascular environment. Perform the power simulation. Flow chamber parallel transparent plastic surfaces Chamber body with (4) fixed using rubber or noricorn gasket (5) (16) consisting of a plastic or glass coverslip (3) mounted on; There is a gap of 114 μm between the two surfaces, and this gap is connected to the inlet manifold (8). Connected population slot (6) and outlet slot connected to outlet manifold (19) (7) (Fig. 11A). with known flow rate and wall shear stress The laminar flow connects to the flow chamber inlet manifold (8) via tube (18). This is achieved by the continuous operation of the pressure pump (2). Figure 11B shows the flowchart The configuration of the member assembly (1) is shown.

Fix the coverslip (3) and chamber body (16) together very tightly. There is a continuous circular groove space (20) around the periphery of the chamber body (16) in order to accommodate the chamber body (16). circle The shaped groove space is covered with a rubber or noricorn rubber gasket with a coverslip (3) on top. It is connected to a vacuum pump by arranging a vent (5). (, therefore, ( 20) by applying a vacuum (21) to the coverslip (3) and the chamber. The main body (1G) is strongly fixed and does not move. In the chamber body (16) Thin inlet and outlet slots open to the inlet and outlet manifolds, respectively. There is. The outlet manifold is connected to a pressure bomb (2). pressure bomb is , can be operated in negative mode or positive mode.

Cells (e.g. endothelial cells) are grown on glass or plastic coverslips (3). grow or various adhesion molecules attach to (3) and tumor cell suspension in culture medium. flows from the inlet Romanifold (16) to the outlet manifold (19). 11th The flow chamber (1) in Figure B is shown upside down for convenience. Cha Place the cell right side up under the inverted microscope stage (Figure 11C) and The flow of tumor cells on the layer (eg, endothelial cell layer) is observed microscopically. tumor cells The observed rolling and stopping pattern (i.e. adhesion pattern) of the video I can record it on tape.

In Figure 11c, cells (9) are grown as a monolayer on a coverslip (3). Alternatively, the adhesion molecules may be attached to the tumor, which is then held in a water bath (17). A laminar flow of cell suspension (I4) passes through the chamber via tube (18). cell The movement of Time difference video cassette recorder using digital image processor (I3) – (11). Adhesion is observed by stopping the cells after rolling. different Shear stress, e.g. 0.4 to 48 dynes/cm", and set time Several fields were recorded (recorded on videotape) to determine the number of cells that bound for 3 min). From karaan [・do (Figure 11 B).

The wall shear stress (T) is defined as 3μQ/2ba2 (where μ=viscosity coefficient, e.g. 1. OcP, Q - volumetric flow jl (cm"/' sec), a - half-channel height, e.g. 5 ．． Calculated as 7xlO' cm and b = channel width, e.g. ], 33 cm. Ru.

Section 1.1. I) Diagram - Through a chamber coated with endothelial cells (9) on the surface Laminar flow of tumor cell suspension (14). "Rolling or arrested cells (15)" Observe with an inverted microscope and record on videotape as described above. arrow, tumor The direction of flow of the cell suspension (14) is shown.

As described above, according to the present invention, a lectin of selectin such as GMP--140 Methods are provided for identifying TACA epitopes to which activity is directed.

Previously, the TACA ubitope was developed using a solid phase coated with activated platelets (e.g. Various Glycos for Adhesion of Blood Cells or Tumor Cells to Plastic Surfaces) Inhibition of sphingolipids (GSL), GSL oligosaccharides or GSL-containing liposomes It was considered based on its effectiveness. In fact, after coating the solid phase with gelatin, activated blood cells are The plate was coated with: Platelets contain GpHb, the major platelet integrin receptor. /111a to gelatin-coated solid phases.

In a study using this method, promyelocytic leukemia 1 (1, 1, Binding of 60 cells is inhibited by liposomes containing Noaron Le' determinants. However, by liposomes containing sialonorbala globoside (SPG) is not inhibited and liposomes containing α1→3 fuconated type 2 chains (Le’) In some cases, it was only slightly suppressed (see Table 1 in Example 3 below). these The results show that Siaron Le' has carbohydrate epitopes confirmed by GMP-140. (Polley et al., Proc., Nat. 1, Acad, Sci, US 88:6224, +991).

However, the above-mentioned method cannot be used unless cell lines expressing only the L-GSL are available. There was a serious limitation that there was no. Myeloid cell line HL60 and monocytic cell line U93 7 expresses exclusively type 2 chains and little, if any, type 1 chains.

On the other hand, all known human tumor cell lines derived from colon cancer, gastric cancer or lung cancer have type 1 chains and 2 Expresses both type chains. For these reasons, the true epitope to which GMP-140 binds is Determining the tope is difficult. To address this issue, a new A method was developed.

GS on fluorescent plastic (e.g. polystyrene) beads (approximately 0.5 μm in diameter) Apply L. GSL is strongly adsorbed to the beads, and therefore specific GS It is known that fluorescent probes containing L can be constructed. Platelets (activated or After incubation with the above GSL-coated Heavey, Measure platelet fluorescence intensity by cytometry.

Using this method, activated platelets are more sensitive to beads coated with relevant GSLs. For fluorescent beads coated with Monon Aron Ru Le″■ (see Figure 3) The bond turned out to be much stronger.

Platelet binding to Noaron Le'-coated beads was determined by anti-GMP-140 model. Inhibited by monoclonal antibody or anti-Noaron Le' monoclonal antibody. However, it was not suppressed by anti-sialosyl-Le''' monoclonal antibody. It was. Binding of activated platelets to Noarosilu Le’-coated beads was observed. However, the level of binding is much higher than that for sialosyl-Le” coated beads. It was low. These results demonstrate that the major epitope identified by GMP-140 The structure shows that it is not sialosyl-Le' but sialon-Le'. There is.

Of course, other epitope structures identified by selectins such as GMP-140 are can be identified using the method of the present invention.

ELAM-I (E-selectin) is expressed on the surface of activated endothelial cells. Ru. ELAM-1 has a carbohydrate-binding domain in the amino-terminal region, In fact, ELAM-1 is known to combine SI-e' and SLe'. There is.

These conclusions indicate that adhesion between tumor cells or leukocytes to activated human ECs is e' suppressed by glycolipids or oligosaccharides, but other test sugars available at the time. This is based on the observation that it was not inhibited by lipids or oligosaccharides ( Pbilljps et al., 5science250:1130, ] I990).

Later, the above-mentioned adhesion was linked to the positional isomer of SLe' glycolipid S L e'' I. (Berg et al., J. Biol. Chem., 266: ] I469.1991; Takada et al., Biochem, B iophys, Res, Commun, 189 Near 13.1991).

The adhesion reaction was said to be inhibited by rgc;8 anti-3Le'mAb (as described above). Ph1llips etc.). However, other related to SLe” and SL e’ There are structural variants and mAbs against those variants, and hypothetical epitope structures. There have been research reports based on the inhibition of selectin-dependent adhesion by

The present invention is the result of a systematic study of selectin-dependent adhesion under static and dynamic environments. It has been accomplished. For example, the methods used include (i) IL-1-activated human Adhesion of tumor cells to umbilical endothelial cells (HUVEC) + (ii) e.g. on E-selectin-coated solid supports by using modified ELAM-1. Tumor cell adhesion; (iii) activated platelets or P-selectin or E-selectin Fluorescence coated with glycoliposomes containing H U V E C expressing Kujin attachment of the particulate solid support; and (iv) transfection with sequences encoding E-selectin. cells coated with glycoliposomes to permanently express E-selectin. Includes N5-1 myeloma cell adhesion.

Therefore, using systems (i), (ii) and (iii), SLe' , SLe' L SLe' IL Lc', Le' and related structures different mAbs; combinations of the above mAbs; noalidases with different substrate specificities or to assess the effect on adhesion of various/alidase and mAb combinations. Ta. Using method (iv), adhesive strength under dynamic conditions was compared.

Specifically, the present invention is characterized by an internal sialon group or a branched structure. Regarding carbohydrates represented by the following formulas (1), (II) and (II) Ru.

Formula (I) has an internal α2→6 allosyl substitution and an α1→4 fucosyl substitution. Concerning type or extension l carriageway.

In formula (I), R3 is H or a noarylic acid residue in the α2→3 bond: RI is a sialic acid residue in H or α2→6 bond: n is 0 or more; and R8 is H or a fucosyl residue in the α1→4 bond.

Formula (II) relates to a type 2 chain structure with internal noarone and fucosyl substitutions.

In formula (II), R7 is as defined for formula I, and R is H or Fucosyl residue in αl → 3 bond, Ri is I], in α2 → 3 bond Sialyl acid residue, NeuAcα2−+8NeuAc or α in α2→3 linkage R s → N e uAc in 2 → 3 bond. In the formula, R6 is /allylic acid It is one or more saccharides other than residues, and n is 0 or more.

Formula (III) relates to a type 2 chain structure, which is a hybrid molecule comprising branches. . Each branch contains an epitope of a single carbohydrate antigen disclosed herein. Become. Therefore, the hybrid molecules used here are not necessarily hybrid molecules. It does not contain all of the two component sugars that contain . Instead, high print comprises an epitope of a component saccharide. Therefore, in the 20th factor, the structure l comprises the epitopes of Le' and SLe', but can be used with various sugars as described below. As is clear from the schematic diagrams of similar structures, all of the Le” or SLe’ molecules are It's not in Brid. Regarding the SLe' part of the hybrid, the intact SLe ``terminal galactose and glycan together with attached fucosyl and sialic acid residues of the molecule.'' Only lucosamine comprises a hybrid. Similarly, regarding Le”, the terminal The only epitope that results in three sugar residues comprises the λibl node. child The epitope used here is the part of the sugar that interacts in the adhesion phenomenon.

In formula III, each of R1° and R11 is galactose, Galβl→4G ]cNAc or Galβ-3GIcNAc, R1 is Gal or Ga 1NAc, and R1 comprises lactosylceramide or an O-linked sugar. Become. Furthermore, R1° and R11 contain fucosyl and sialylic acid residues. I can do it. The 7% hybrid structures are characterized by their respective epitopes contained in them. Identified by Therefore, structure 1 in FIG. Le’/SLe” is displayed.

Formula I exclusively contains type 1 chains, namely Galβl→3G]cNAcβ1→3GaI, and of tumor cells (e.g. Co1o201 cells) expressing repeats of and substitutions thereof. Various mAbs and noalidases and their combinations of E-selectin-dependent adhesion It is based on suppression through coercion. E-selectin dependence of Co1o201 cells Adhesion was minimally inhibited by mAb CA19-9 (against SLe''), but mAb FH7 (against diallosyl Le″ and monocyalone Le″ II) was moderately suppressed. Co1o201 adhesion was performed using mAb CA3F4 (mono-alloy). ) or combination of CA19-9+CA3F4 It was most strongly suppressed by

Specific reactivity of FH7 with diallosyl Le' and mononoarone Le'll The specific reactivity of CA3F4 and monoalonor L e″II has already been described. (Nudelman et al., J. Biol. Chem., 261:5 487.198G).

The epitope structure was further clarified based on the following considerations. Co1 o201 cells to NeuAcα2-+30a! (R in formula I) is cleaved Treatment with New Cavus disease virus (NDV) noalidase results in E-sere Cutin-dependent adhesion was slightly suppressed, but Arthrobacter ureafaciens (also designated AU or AV in the drawings) or Vibrio cholerae (VC) Noalidase (both NeuAcα2-6 binding to GlcNAc or Gal) Such adhesion can be removed by treatment with completely suppressed. Therefore, the internal 2→6 noaron structure is involved in adhesion. It is clear that there are. NDV sialidase is mAbcA19-9 or CA In combination with 3F4, adhesion was strongly suppressed. The above results are herein were obtained in both the static and dynamic systems described.

However, it becomes clear in dynamic flow systems that more closely simulate physiological conditions. As shown, the binding kinetics of selectins are vibrant, i.e., e.g. White blood cells or tumor cells move with considerable velocity in large unoccluded vessels; and can move at slower speeds in tissue space. Under static conditions, cells interact Action involves interaction with a first set of molecules that share common properties and is non-static. Under normal conditions, cellular interactions differ from those shared by the first set of molecules. interaction with a second set of molecules that share common properties. moreover, Under non-static conditions, binding requirements vary depending on the speed at which the cells are moving.

For example, E-selectin (ELAM)-mediated adhesion of HL60 cells is either static or static. or when cells react in a slow moving setting, or when cells react in a fast moving setting. It depends on the carbohydrate structure.

Under static or low shear conditions, ELAM induces α2→3 no-allylation and binds preferentially to α1-3 fuconolated structures, whereas under high shear conditions ELAM has other structures such as Le', Le', H, and various high structures such as Le'/SLe''. preferentially binds to hybrid structures.

Formula 11 shows that 2 type chain only, i.e. Galβ1→4GlcNAcβl−+:3Gal and its repeats 1 (E-selectin-dependent adhesion of L60 tumor cells) It is based on the suppression of HL60 cells were treated with NeuAcα2-+3Gal (formula When treated with NDV alidase, which cleaves R, in II, cells are Although it remained strongly attached to the lectin-coated plate and activated EC, Reactivity with anti-8Le' mAb completely disappeared. to E-selectin or EC To completely suppress adhesion of GIcNAc or Ga1, NeuAcα 2−+6 (i.e., in addition to Neu, A cα2→6 shown in formula II, Treatment with AU or VC noalidase, which cleaves R2), was required. The R6 group is , is easily cleaved by AU and VC soalidase, but is easily cleaved by NDV soalidase. Therefore, it will not be cleaved.

For Formula II, various mAbs affecting E-selectin-dependent HL60 cell adhesion This became clearer by observing the effects of

Adhesion was achieved using a combination of NDV alidase and anti-Le' mAbsHl or anti- -8Le' was strongly inhibited by the combination of mAb SNH4 and SHI.

The relevance of compounds of structure III was determined by E-selectivity of tumor cells expressing type 2 chain saccharides. A novel approach using various mAbs and noalidase for ion-dependent adhesion E-selectin transfection into coliposomes was also used to test N5-1 cell adhesion.

For example, NDV analysis of H I--60 cells that removes NeuAcα2-6Gal. Cells adhere to E-selectin plates and activated endothelial cells by lidase treatment However, the reactivity between the cells and the anti-3Le' mAb completely disappeared.

Adhesion was effectively inhibited by a combination of mAbs against Le' and SLe'.

The internal sialonyl structure is different from the known type 1 structure (Nudelman et al. above). Therefore, a type 2 chain structure with an internal noarylic acid residue was unknown until now.

The data presented in this application demonstrate the natural occurrence of shrimp I.-B.

A structure capable of binding to El and AM-1 can be synthesized using known techniques. for example, Carbohydrates can be chemically synthesized using known and commercially available reagents, or can be synthesized using known and commercially available reagents. It can be synthesized by performing appropriate conjugation using a capable enzyme. For example, the well-known Noah Inducing the basic backbone of carbohydrates using lonl transferase and fucon rutransferase be able to.

In addition, carbohydrates that can bind to ELAM-1 can be detected using ELAM-1 as an adsorbent. It can be isolated and isolated. For example, purified ELAM-1, cells expressing ELAM-1 or can use membrane assays of cells expressing ELAM-1. ELAMI inert bee It can be immobilized on a solid phase such as a matrix or the inner wall of a container to improve separation performance. Wear. Next, HL60 or Co1o201 cells obtained by a known method were A suitable carbohydrate capable of binding to ELAM I, such as an extract, is added to ELA, M-1 affinity Exposure to 7 Tricks. Wash to remove unwanted and non-specifically bound components. After removal, ELAM-1 and carbohydrates capable of binding to it are harvested together. EL Carbohydrates bound to AM-1 can be removed from ELAM-1 by e.g. Separate and collect by changing the temperature. Chromatograph various carbohydrate species It can be identified using a known procedure such as

In addition, cells known to mainly express type 1 chain structure or type 2 chain structure are grown. From there, a membrane sample is obtained by a known method. Glycolipids and glycoproteins in membrane samples Fractions were obtained using known techniques and applied to an affinity column as described herein. Antibodies against carbohydrate epitopes such as to form an affinity matrix. affinity chroma In the tography operation, further known methods such as HLPC and TLC are used. The bound substances are eluted and separated.

T1. When using C, the molecules separated in the separation medium are labeled and tagged. can be exposed to ELAM-1-expressing cells that play the role of In other words, ``cells are radioactive Can be metabolically labeled using isotopes. ELAM-1 expressing cells were isolated from EL AIVI-1 epitopes are bound to each position of the separation medium where they are found.

The location of cell binding sites was determined by autoradiography of the TLC matrix. and identify the ELAM-1 epitope-carrying molecule. TLC matrix Each part can be excised and molecules extracted.

As mentioned above, carbohydrates of formulas I and 1 can be derived to have more desirable therapeutic properties. It is possible to provide oligosaccharides having Therefore, for structures comprising formula I or II, A portion can be replaced, for example, with a sulfur-containing sugar or a fluorine-containing sugar. oligosaccharide The derivative is incorporated as a fucose residue in formula I or II in place of the natural component. Using a suitable reagent containing a modified substituent such as 6-trifluorofucosyl and can be prepared using the methods disclosed above.

Carbohydrates capable of binding to ELAM-1 can be used as immunizing antigens to target ELAM-1. Antibodies capable of binding to a carbohydrate capable of binding can be obtained. The method explained above and documents cited herein (incorporated by reference). polyclonal antibodies or monoclonal antibodies using methods such as those described in can generate null antibodies. Monoclonal antibodies are preferred.

Since ELAM-1 may play a role in mediating cell-cell interactions, ELAM-1 A. Blocking the binding between M-1 and carbohydrates that can bind to it may be beneficial. . Thus, for example, carbohydrates capable of binding to ELAM-1, ELAM-1, Antibodies against ELAM-1 or carbohydrates capable of binding to EL A~1-1 Binding between ELAM-1 and carbohydrates capable of binding to it using antibodies that can be blocked. Carbohydrates capable of binding to ELAlvl-1, ELAM-1, EL Antibodies against AM-1 or carbohydrates that can bind to El, AM = '1 Therapeutic antibodies can be easily and routinely used in the pharmaceutical field in therapeutically effective amounts. It is administered via a well-defined route.

As mentioned for formulas (1) and (II), the terminal sialic acid is ELAIVI -] is not essential for carbohydrates that can bind to. Key elements held in common are terminal galactose, glucosamine, α2→6 sialic acid, and fucose residues.

Therefore, antibodies capable of binding to such structures may be able to bind ELA~1-1-mediated interactions. effective in suppressing Suitable antibodies are CA3FA and FH7.

Compounds of formula (II1) in which the relevant epitope comprises a branched structure, e.g. Le'/SLe' has high affinity for ELAM-1 under high shear window J conditions. It was clearly confirmed that the protein contained a binding site. However, such The structure is simple S1 for ELAM-1 under low shear stress conditions or static conditions; The binding capacity may be smaller than e'. When using a hybrid, one Terminal galactose α1→3-linked fucose and others to branched Glc'NAC α2→3-linked sialylic acid and α1→3-linked fucose in one branch are hybridized. This is an important part of the structure. Therefore, Le' as well as 5H--1 and FH-2 Antibodies capable of binding to SLe' such as PH-6, 5NH-4 and 5NH-3 have high Together they are effective in suppressing ELAM-1-mediated adhesion under shear stress conditions. Ru.

A number of epitopes recognized by ELAM and GMPI40 are O-linked glycans. Selectin-dependent cell adhesion is promoted by inhibiting O-glycolylation. (Kojima et al., Biochem, Biophys, Res, C ommun, 182:1288.1992). Therefore, formula (1), (I Preferably, the compounds of I) and (III) are supported on a 0-linked carbohydrate chain. There is.

E. A further implication of blocking LAM-1-mediated interactions is that carbohydrate antibodies or combinations of antibodies to inhibit ELAt-1 binding to relevant carbohydrates. It is to interfere. The carbohydrate or antibody is capable of binding to ELAM-1 or related to carbohydrates and, in some circumstances, appears to bind specifically to ELAM-1 It can be a carbohydrate or an antibody that is not a carbohydrate. For example, SLe'' and Le' The combination of antibodies against ELAM-1 is only effective in inhibiting the ELAM-1 interaction. Ru. Suitable SLe' antibodies are SN H3 and SN H4; Le' antibodies are SHI and FH2. Those skilled in the art will appreciate the specific examples described below. The methods taught herein using the reagents disclosed herein, while taking into account the Other suitable combinations can be determined to implement the method.

The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples. do not have.

Example Example 1 Synthesis of lactose derivatives A, methyl β-D-lactone Heptaacetyllactone imide h (Z imme rmann et al., J, C arbohydr, Chem, 7:435, I988) by standard methods. Dry dichloromethane containing trimethylrinyltrifluoromethanesulfonate (Grundler & Schmit, Lie). bigs, Ann, Chem, 1984:l826.1984). silica gel Purify by column chromatography (toluene/EtOAc = 1.1 (volume ratio)) After production, it was de-O-acetylated with 0.0 IM sodium methoxide to form imidate. Methyl β-D-lactosites were obtained in a yield of 68%: melting point 211-212″C ( Literature value 205°C, Sm1th & van C1eveSJ, Am, Chem , Sac, 77:3159, 1', c5.01H20), ibid.

B, phenyl β-D-thiolactosite Lactose octaacetate (Hudson & Kunz, J.

Am, Chem, Soc, 47:2052.1926) in dichloromethane at 0 °C. thiophenol and 5nC1, (Nicolaou et al., J. Am, Chem, Soc, 110nia 910.1.988) Ruheptaacetyl β-D-thiolactosite was obtained in a yield of 80%. The product, Me Deacetylated with NaOMe in OH and Amberlyst® )15. BioGel® column using water as eluent After purification of the product, lyophilization of the sugar-containing fraction was performed to obtain phenyl β-D-thio Lactocytes were obtained as a white amorphous powder.

C, lacto-N-tetrose Oligosaccharide (Galβl-3GIcNAcβ1→3Galβl→4Glc) BioGelP-2 from milk was pretreated with ethanol and using water as eluent. After repeating column chromatography, reverse phase (C,) high pressure liquid chromatography using water tography (Dua & Bush, Ana1.Biochem, 133: 1983). ’)I-NMR spectra of the authentic sample (BioCa Overlaid with those from rbChemi ca Is, Lund, Sweden). .

The polyethylene glycol derivative reaction scheme of D, β-D-lactone is shown below. show.

the law of nature The polyethylene glycol derivative of β-D-lactone can be easily Amino group 2 (Za) 1psky etc., Eur, Pol acetyl-〇-β-D -galactopyranosyl)=(1→4)-2゜3.6--tri〇-acetyl-β -D-glucopyranoside l and polyethylene glycol methyl ether (average Molecular weight 2000; Aldrich Chemical, Milwaukee, Wis. Key). l (100 mg, O, l 2 mmol l) and 2 (16 3 mg, 0.082 mmo]) was dissolved in dry N, N-trimethylformamide (2 ml) at room temperature for 2 hours to obtain LH- After chromatography at Obtained with a yield of 91%: [α), -5.3° (cO,5, chloroform). continue, After saponifying 3 with 0.05M sodium hydroxide at room temperature for 1 hour, it was lyophilized and quantified. The desired lactone compound 4 was obtained: [α) D-2,4° (cl, 0, chlorophosate) Lum).

Example 2 Effects of lactose and lactose derivatives on metastasis of BI6 melanoma cells The highly metastatic BL6 clone of the BI6 melanoma cell line was initially developed by Jean Dr. 5tarkey (Montana 5t located in Bozeman, MO) ate Unv,) and was metastatically induced in syngeneic 057B1 mice. I re-selected it. C57B1 mice were maintained in plastic canes under a filtered air atmosphere. The animals were fed water and food pellets ad libitum. Cells were treated with 2mM glutamine and 10% RPM II 640 supplemented with beef tallow serum (Fe2) and cultured with 2mM EDTA. It was separated with phosphate buffered saline (PBS).

Viability was estimated by trypan blue exclusion test.

A suspension of BL6 cells (1 to 3xlo'' cells/ml RPMI 640 medium) Prepare and aliquots of various oligosaccharides at different concentrations for 5-10 min at 37 °C. were incubated in the presence or absence of. After incubation, typical Generally, 3xlO' or 2xlO1 cells (with or without oligosaccharide pretreatment) / 200 μl was injected via the tail vein into 8 week old female mice. After 18-21 days, The mice were killed and the lungs were fixed in 10% formaldehyde in PBS (pH 7,4). and by counting tumor cell colonies under a dissecting microscope under the above conditions. Background values of the number of metastatic melanoma colonies in the lungs of patients were obtained. colony The data on the number and size of the samples were statistically processed using the analysis of variance (ANOVA) method. 1 Colonies with a diameter of 1 mm or more are considered large size, and colonies with a diameter of less than 1 mm are considered large size. were considered small size.

In one experiment, BL6 cells were treated with various concentrations of lactose, lacto-N-tetro (Galβl→3GIcNAcβl→3Galβ1→4GLc), methyl β -D-lactosite or phenyl β-D-thiolactosite for various times. Cubated. In the majority of experiments, a concentration of 0, IM was used and cells were grown at 37°. Incubate for IO minutes at C and gently centrifuge for 10 minutes at 400 separated from containing medium, resuspended in RPMI 1640, and injected via the tail vein ( 3 x IO' cells in 0.2 ml suspension). In some experiments, 2xlO' Cells were injected and colonies were counted on the 21st day. Ink in various sugar solutions The viability and cell growth ability of BL6 cells after activation were evaluated using trypan blue exclusion assay. experiments, in vitro RPM11640 plating under normal conditions as well as age-matched C5 Tested by testing tumor growth upon subcutaneous inoculation in 7B1 mice.

BL6 cells were preincubated with lactose and lacto-N-tetrose. When the cells were injected intravenously under the same conditions after lactose and lactose-N-tet loin reduced metastatic colonies in the lungs by 26% and 36%, respectively. . BL6 cells were grown at 0.0% under the same conditions as above. IM, 0. OIM or 0.005M Methi The number of metastatic lung colonies increased compared to the control when treated with Le β-Riichi Rough decreased by 43%, 16% and 8%, respectively. O,1,M methyl β-D-lac We performed three separate experiments to demonstrate the significant decrease produced by the It was found to be between 5 and 45%.

In a second independent series of experiments, methyl β-D-lactosodo or phenyl β-D - each under different conditions following pre-incubation with thiolactocytes. upon treatment with methyl β-D-lac I-cyto or phenyl β-D-thiolactone. Therefore, metastatic colonization, i.e., total colony number is 35-50% of the control value. Diminished. The decrease in large colonies was greater than the decrease in small colonies in all experiments. (Fig. 1). ). Both methyl β-D-lactonodo and phenyl β-D-thiolactonodo, It showed a slight promoting effect on cell number increase and thymidine uptake in vitro. death Therefore, the inhibitory effect on tumor attachment may be due to the inhibitory effect on cell growth in vivo or in vitro. It has nothing to do with the effect of

In separate experiments, the effects of methyl β-reactin on melanoma cell metastases The effect was measured by incubating the oligosaccharide with tumor cells after administration. concrete 1 ml of methyl β-D-lactone (a degree 0.25M or 0.5M) was injected intraperitoneally into mice. After 10 minutes, BI6 melanoma cells were injected intravenously. . Lung colonies were counted after 19 days. Methyl β- before inoculating tumor cells Injection of D-lactonod significantly reduced the formation of lung metastatic colonies. (Figure 2).

In separate experiments, mouse melanoma BI6 mutants (BL6/ FIO/Fl/Wa4) has already been identified as a melanoma-related antigen. GM3 gangliondo (Hirabayasl et al., J, Bioi Chem , 260:13328.1985; Nores et al., J. Immunol, 13 9:3171.1987) were expressed to the same extent. GM3 is highly expressed in endothelial cells It interacts with L a c Ce r expressed in . La　cCe　r coating solid phase or The degree of adhesion of the B16 mutant to endothelial cells was also similar to that of metastatic (MP) cells. there were. In contrast, integrin-dependent adhesion of the 816 mutant It was almost the same as FIO and Fl (see Figure 4).

These are the BI6 adhesion of LacCer, the molecule GM 3-L ac Cer This suggests that it is based on interaction. In addition, 816 mechanisms against endothelial cells Lanoma adhesion is not only methyl-β-lactone but also Laccer lipo It is also inhibited by Gg3Cer liposomes, Gg3Cer liposomes, and GM3 liposomes. (See Figure 5).

Furthermore, the above-mentioned observation on the metastasis-inhibiting effect of methyl-β-lactone was reported separately. The patient was given injections of methyl-β-lactone. That is, tumor cells are transferred intravenously. After the injection, methyl-β-lactone was injected intraperitoneally. In these experiments, 0. Lung metastatic colonies were isolated by injection of 25-0.5 M methyl-β-lactosod. The number decreased by 40-70% (see Figure 6; A = PBS control, B = 0.25 MMC -β-Lactondo; C=0.5MMe-β-Lactondo, D-05M Lactose ;E=0.25MN-acetyllactosamine;F-O,5MMe-β-galacto /do, intraperitoneal injection).

Capillary endothelial cells are treated with antibodies against H/Le'/Leb such as antibody MIA-15-5. It was highly reactive with the body. This observation shows that Ulex Europe, I Previously reported observations of endothelial cell staining (Holthofer et al. , Lab, Invest.

45+391.1981; 47:60.1982).

Liposomes comprising H-1 or Le' were prepared and treated with various glycolipids at a range of concentrations. was exposed to a fixed plate.

As shown in FIG. 7, the H-loaded liposomes were bonded to e'. On the other hand, Le'-carrying liposomes only bind to H-coated plates. It turns out that it does. H and balagloboside are related, and the difference is that H has a terminal fuco the presence of a -base residue.

Therefore, 4, cells expressing Le' or Le' are endothelial cells expressing I]. It can also adhere to cells and perhaps Le'. These interactions may There is a first step in which cells adhere to endothelial cells.

Example 3 Expression of noarosyl dimer Le' and metastatic KUM-LK-2 in human lung adenocarcinoma cell lines , a human non-adenocarcinoma cell line characterized by spontaneous lung metastasis in nude mice. . After screening 35 human cancer cell lines growing in nude mice, Only metastatic adherent cell lines in the lungs of infected mice were obtained. KUM-LK-2 as the parental cell line were used to obtain sub-cell lines that could be injected IV to produce lung metastases by limiting dilution. .

Limit dilution was performed as follows. K　U　　　-L　K-2　, 10%FC RI” MI1640 supplemented with 3 (manufactured by Hyclone, Logan, Utah) 5% Co Cultured at t/3700 in a 95% air atmosphere. 2mM EDTA solution Briefly processed, washed twice in RPMII640, and washed in RPMI containing 10% FC5. A single cell suspension was prepared. Cell survival measured by trypan blue exclusion staining The degree was >98%. The cell suspension containing I cells per 100 μm was Corning, NY in 6-well microtiter plates. ng Glass Works! Transfer to 8 wells and culture continuously for 24 hours. Ta. Next, each well was observed using a phase contrast microscope.

Three cell lines (HAL-8, HAL-24 and and HAL-33) in 25 cells obtained by limiting dilution based on stable cell morphology. Selected from clones. The 25 clones showed variation in cell growth in vitro. A total of 63 clones were selected from 63 clones that showed not only no cellulosis but also a stable morphology.

All clones developed spontaneous lung metastases. However, 1. V.

After injection, clear differences between the clones were observed in lung metastasis deposit formation. high 2 clones with MP, 5 clones with low MP and no MP. Eight clones were identified.

The selection was repeated by IV and injection of clones to select the safest one with consistent MP. A stable sub-cell line was established. These are the same for nu/nU mouse lungs. HAL-8, HAL-33 and HAL with high MP, low MP and no MP respectively. 24 (see Table 1 below). From visual and microscopic examination, three sub-details are detected. It was determined that none of the cell lines showed metastasis to other organs or lymph nodes. sub The cell lines represent stable variants that originally existed in KUM-LK-2. Chromosomal portion Analysis confirmed that each subclone was distinct.

Metastatic potential of HAL-8, HAL-24 and HAL-33 in "nude mice" +1AL-81515, 8 (8-23) 22 15.0 (10-22) 46 16.3 (II-25) HAL-24150 ) IAL-33154, 3 (3-7) 22 5.1 (2-8) 46 5.8 (3-8) ゛, Nude mice were injected via the tail vein (2xl O' cells). 56 days after injection, mice were sacrificed and metastatic nodules on the lung surface were examined under autopsy. Counted with a microscope.

5 ・Average of 6 animals (range within 0) Cell surface expression of various carbohydrate epitopes was determined by Ronru Le’ (mAb 5NH4), Sialonru dimer Le’ (mA b FH6), T (mAb HH8), Tn (mAb IE3) and Cia Various monoclonal antibodies (mA b) was analyzed by cytofluorometry. All antibodies used were The culture supernatant from each hybridoma was diluted with 10 μg/ml immunoglobulin. It was adjusted accordingly. Siaron Le' (Structure l), Siaron Le' -Le' (Structure 2), Dimer -Le'' (Structure 3), Trifucosyl Le' (Structure 4), Le' (Structure 5), H (Structure 6), 5A-Le' I (Structure 7) ), 5A-Tn (Structure 8), Nosialon Le' (Structure 9), Monosialosyl -Le’ll (Structure 10), GM3 (Structure 11), 5-PC (Structure 12), Le The structures of "(Structure 13)" and "Le" (Structure 14) are shown below. R is the carrier portion Represents a child.

Structure, AU2 1st shift and F’uC61FuCQ1 Structure】Eye= 1 drip Torture U NeuAca2-*3Galβ1 → 4GlcNAcβ1 → KoGalβl → R 豫I And- 1xlo' cells were prepared for each mA,b treatment. cells , mAb for 1 h incubation at 4 °C, RPMI 164 (Washed 1'2x. Next, use goat anti-mouse IgG or IgG 50x in PBS. M-FITC (Boehrini located in Indianapolis, Indiana) ge r-Mannhe im) and incubate at 4'C for 30 minutes. It was done. Finally, cells were washed three times, resuspended in PBS, and EPMC3PRO FILE flow cytometer (manufactured by EpicS, Hialeah, Florida) ) was analyzed. The experiment was repeated using three different cell generations.

Six types of carbohydrates for sub-cell lines HAL-8, HAL-24 and HAL-33 The expression pattern of the biological epitope (confirmed by the respective mAb) is Almost identical contours (for the three subcell systems) except for the case of the dimer Le” protein contour). In particular, HAL-8, HAL-24 and HAL-3 3 highly and equally expresses sialon-el and sialosyl-Tn structures. It has been found. Each of the three lines expressed small amounts of Le' and Tn, but ■ It didn't appear. In contrast, expression of the sialon-dimeric Le1 was high, HAL-33 was moderate, and HAL-24 was low.

Release of sialosyl residues was evaluated as follows. Cells in 2mM PBS Separated using DTA, washed and resuspended in 9 volumes of PBS. 1m cell suspension 1 and 0.1 of Mycobacterium perfringens alidase. 20/ml (Type X, Center, Missouri) 376C along with Sigma Chemical Co. (manufactured by Sigma Chemical Co.) Incubated for 5 minutes. After incubation, cells were washed three times and R Resuspend in PM I 1640 and test for expression of MP and Sialon-L dimer Le'. We investigated. The MPs of HAL-8 and HAL-33 are effective for noalidase treatment of cells. more completely inhibited (see Table 2 below). The expression of the sialon Le dimer Le’ is It is thought that it plays an important role in blood circulation.

Table 2 Effect of sialidase treatment on the metastatic properties of clones HAL-8 and HAL-33'' sound Control (PBS) 1 (AL-816,3 (9-24) HAL-334,6 (3-7) Sialidase HAL-80 HAL-330 “　: Nude mice were injected via the tail vein (2xl O' cells). 56 days after injection, mice were sacrificed and metastatic nodules on the lung surface were examined under autopsy. Counted with a microscope.

1: Average of 6 animals (range within 0) Example 4 Identification of carbohydrate epitopes that can bind to the lectin domain of GMP-140 Obtained from Oregon Red Cross (Boatland, Oregon) Platelets were isolated from "platelet-rich plasma." Remove contaminated red blood cells by 80x The cells were removed by centrifugation at g for 10 minutes. Centrifuge the platelets at 300xg for 10 minutes. , 22mM citrate buffer containing 0.35% bovine serum albumin (BSA). and suspended in Tyrode's buffer (pH 6.5). Platelet suspension (lxlO”/m 1) was incubated after addition of thrombin (final concentration: IU/ml). (pH 7,2,376C for 15 minutes). The mixture was then heated at 37°C without stirring. and incubated for 10 minutes. Thrombin-activated platelets were collected in phosphate-buffered form. Fixed with an equal volume of 2% formaldehyde in saline (PBS), pH 7.2, Washed twice with PBS containing 1% BSA. Activated platelets (not non-activated platelets) ) was 2.5 μg/ml anti- GMP-140mAb AC1,2 (isotype IgG+, California Manufactured by Beckton-Dickinson, San Jose, State) showed his sexuality. Fluorescently labeled goat anti-mouse Ig (Burlin, CA) was then used. Tag in the game.

Co., Ltd.) was reacted with 50 μl. Flow of activated platelets using mabAcI,2 The relationship between the cytometry contour and the cytometry of non-activated platelets is shown in Figures 8A to 8A. Shown in Figure 8D.

Activated and non-activated platelets were incubated in Ca''+-free PBS, pH 7.2. The cells were fixed with rose formaldehyde and washed twice with Ca'-containing PBS containing 1% BSA. The platelet count was determined by resuspending in Ca'-PBS with 1% BSA and 0.1% azide. The concentration was adjusted to approximately 1×lO′ cells/ml. The cell suspension was stored at 4'C and within 24 hours Binding assays were performed.

Fluorescent polystyrene latex beads are manufactured by Mo1ec in Negeen, Oregon. Obtained from ular Probe. The beads are approximately in diameter with sulfate groups on the surface. They were yellow-green fluorescent beads of 0.5 μm (actual diameter 0.488 μm). ETOH3 Add beads (lxlO') to 0μl and add GSLIOμg to 200μl of C=M. solution, mixed well and dried under a stream of N2. Dilute the residue with ethanol 2 00 μm, briefly sonicated, and dried under a stream of N2.

The dry residue was diluted with 3% BSA and 0.5% BSA. 1% azide-containing Ca''-PBS 2ml Resuspend, sonicate for 10 minutes, let stand at 37°C for ε0 minutes, and remove beads with BSA. blocked the surface. Centrifuge the suspension at 3000xg for 10 minutes and transfer the suspension to a bead base. The cells were washed twice with Ca''-PBS containing 1% BSA and azide, and finally Then, the cells were suspended in 500 μm of the same medium and stored at 4°C.

Platelets (inactive) containing approximately 2xlO' platelets were fixed with paraformaldehyde. Fluorescent G containing approximately 2 x 10' beads of 20 μm suspension (activation) Mixed with 10 μm of S L coated beads, and after thorough mixing, was left standing at 37'C for 30 minutes. . The platelet suspension was mixed with 200 μl of Ca'--PBS and subjected to flow cytometry. Lee (EPICS Profile, Coulter Cytometry, Hire, FL) Analyzed by Leah).

Contains most of the signal produced by platelets and excludes the signal produced by free beads. In order to set the gate for removal, flow cycles of platelets alone and beads alone were performed. Tometry analysis was performed. Binding index (Bl) with fluorescent GSL coated beads The average fluorescence intensity (Mfl) of the incubated platelets was determined by applying fluorescent valve GSL ( Control) The value divided by the MFI of platelets incubated with beads. It was calculated by Bl values for various GSLs are shown in Table 3 and Figure 9. 9th In the figure, hunted bars represent non-activated platelets and oven bars represent activated platelets. Represents platelets. 5A-Le’, 5A-Le’, SPG, GM3 and Le” Binding index of activated platelets (Bl) and binding index of non-activated platelets (BI) The ratio between the two is also shown in the "Ratio (Activity/Inactivity)" column.

Table 3 G S L rhombin activity on L-coated sulfate-containing polystyrene beads binding index of platelets GM3 1.0±0. IO,7±0.5SA-1,e’4.2±1.0 4.2±0.5SA-Le' 8.1±1.06.　I±0.2SPG　O , 8±0.2 0.7±0.21, e''' 1.l±0.3 1.0±0.3'' Values are the average of 4 separate experiments.

The mAb did affect platelet binding to fluorescent GSL-coated beads. small blood The plate was coated with anti-〇MP-], 40mAb l0P62 (available in Marseille, France). (manufactured by ImmunoteCh) at 37°C for 30 minutes. binding assay was performed using GSL-coated beads as described above. Contrastive conclusion Non-specific mouse IgG (to μg/ml) was used for the assay.

In addition, anti-5A-Le' coated beads (2xlO' pieces) of 10 μm were added to the anti-5A-Le' mAbCA 19-9 (7US IgG+; Signet Laboratories) (20μg/ml) with 20μI Both were incubated for 60 minutes at room temperature and used for platelet binding assay. . Anti-8A-Le'mAb SNH4 and non-specific mouse IgG were used as controls. did.

The results are shown in Figure 1O. In Figure 10, the horizontal axis represents the inhibition rate, and column l is Represents anti-GMP-140 mAb l0P62, column 2 represents anti-3A-Le' mAbcAl　9-19　(Alternative mAbs are NKHI and NKH2) Column 3 is anti-8A-Le” mAb SNH4 and column 4 is normal mouse. Represents sIgG.

Activated platelets are susceptible to GMP as evidenced by high reactivity with anti-CD62 mAb. -140 was highly expressed. (Figures 8A to 8D) Expressing GMP-140 Activated platelets exhibit high binding to fluorescent beads coated with SA-Le. (See Figure 9).

Platelet binding to beads coated with 5A-Le' was observed; The extent was much smaller than in the case of “e” (Figure 9). No binding was observed for beads. Furthermore, 5A-Le’ coated beads Platelet binding to anti-○MP-140 mAb and anti-8A-Le'm Ab or not inhibited by anti-8A-Le'mAb (Figure 1O).

Example 5 Interleukin-1 activated human venous endothelial cells in a dynamic flow system Effect of various monoclonal antibodies on adhesion of human colon cancer Co1o205 1st. Adhesion was measured using the dynamic flow experimental system shown in Figure +A to Figure 1 ID. Established. 3 minutes at different shear stresses, e.g. 0.4-4.8 dynes/cm' From several fields recorded on videotape, the number of cells that joined during the I counted. The viscosity coefficient is 1. OF, half channel height is 5.7x, IO '-'' cm, and the channel width was 1.:3 cm.

This system has been used to target various human tumors and various tumor-associated carbohydrate antigens. We investigated monoclonal antibodies. Human colon cancer C on activated human endothelial cells The results of one study regarding the adhesion of o1o205 cells are shown in FIG. 12. Figure 12 In , the horizontal axis represents wall shear stress (dynes/cm').1. The vertical axis is cell adhesion (x 1O-27 field).

In prisoner 12, the meanings of the symbols are as follows. White circle, unrelated mouse Ig C; mixture of +IgM (control), black triangles, mono-allosilyl against Le''I Monoclonal antibody CAl9-1 white triangle, monochrome against Noalonoru Le' Null antibody 5NH4; black circle, monoaloneru L　e''　II and dinoaloneru - Monoclonal antibody FH7 against Lc', and black square, unrelated mouse I gG+ l Mixture of gM and non-activated endothelial cells.

The results showed that Co1o205 adhesion to activated endothelial cells was enhanced by antibody FH7 (particularly is most strongly suppressed by high wall shear stress (5-10 dynes/cm'). I understand that In contrast, antibody C/'119-9 did not show any suppressive effect. won. These findings suggest that tumor cell adhesion to endothelial cells is a monoalone rule. Le’ll or Gin Aron Roux L e’ and Inotaleukin-1 Activation Sele This suggests that the process may proceed through interaction with cutin.

Example 6 Selectin-dependent adhesion of HL60 cells HUVEC (manufactured by Cernoste 1, Kirkland, Wanonton), 48 With a well plate (manufactured by Kostar, Cambridge, Mass.), The cells were cultured until surface growth and stimulated with IU/ml L-1 for 4 hours. Non-irritating 1 (UV EC was used as a control.

E-selecdin (ELAM-1) for TL--1 stimulus HU VEC Expression was determined by reactivity with anti-E-selectin mAb 3B7 (IgG, J). confirmed (Graber et al. 1.J, 1mm, 145:819, +990). ]( L60 and Co1o201 cells were pretreated with glycotylation modifiers. After being metabolically labeled by incubation in the presence of [′H]-thymidine, HU　V　E　 C was added to the coated plate.

After 15 minutes of incubation, the plate was washed with PBS and the number of adherent cells was determined. Estimated by converting from radioactivity counts. In another series of experiments, a 96-well play 1. The transmen is manufactured by Falcon Corporation in Suncoln, New Jersey. Truncated recombinant E-selectin lacking the brain and cytoplasmic domains (clear Water, etc., Nature349 Near 99.1991) 0.1-jμg/ml, 18 Applied for hours. Next, the plate was coated with I%BSA, washed with PBS, and The metabolically labeled glycolylation-modified cells were plated. 60 minute ink wash After cleaning, wash the notebook with PBS and calculate the number of adherent cells from the radioactivity count. It was estimated by

Cell adhesion to activated or native platelets plated and fixed in 48-well plates The assay was performed in a manner similar to that described above (Handa et al., Biochemis try30:] I168.1991).

F(L60 cells were pretreated with 2mM Benzyl-GalNAc for 72 hours. , labeled with [3H]thymidine. After washing with PBS, 1x106 cells were added to each well. The plate was incubated for 30 minutes at room temperature. Washed and unwashed After removing the bound cells, separate the bound cells with trypsin and add liquid non-chloride. Calan l-1 was determined by the counter. Platelets bound to the plate were treated with anti-P- Selectin mAb l OP-62 (+2, l:6 dilution) (France) (manufactured by Immunotech, Marseille) for 30 minutes at room temperature. After fermentation, HL60 cells were added to determine the dependence of adhesion on P-selectin expression. The gender was evaluated. Non-specific mouse IgG was used as a control.

Adhesion assay in dynamic flow systems Parallel plate laminar flow chamber with injection pump (model 935, Cambrino, MA) (manufactured by Harvard Apparatus) located in the , the flow shear stress present in the physiological microvascular environment has been investigated. flow The chamber is made of parallel transparent plastic using Silasty and Kugom caskets. It is composed of a glass plate with fixed surfaces, and there is a gap of 114 μm between the two surfaces. , and this gap is connected to the inlet and outlet.

A laminar flow with a predetermined velocity and wall shear stress is introduced into the inlet connected to the inlet of the flow chamber. This is accomplished by operating an inlet pump. Growing EC as a single layer on a glass plate or apply adhesion molecules and pass a laminar flow of the cell suspension through the chamber. Ru.

Cell movements were observed using a reversed-phase contrast microscope (Diaphot-TMD manufactured by Kon). Observe and record with a time difference video cassette recorder. Stop after rolling cells and observe adhesion. At different shear stresses (e.g. 0.4-4.8 dynes/ c m2 or 0.76-155 dyne/cm') number of cells bound in 3 minutes, Counts were taken from several fields recorded on videotape. Wall shear stress ( T) was calculated using the formula L a w r e n c e etc. (B]ood7 5:227.1990) T = 3μQ/2ba' (In the formula, μ - viscosity coefficient (+, 0 cP), Q - volumetric flow rate (cm'/sec), a - half channel height (5.7xlQ' for the experiments described here), and the b-channel height channel width (1.3 cm)).

HL60 adhesion to E-selectin coated plates under static conditions. Cell line 1-IL60 was tested using a specific mAb (Symlngton et al., J. Irnm Unol, 134:2498, l 985), type 2 chains and Noah It was found that only lonlylated/fuconlylated derivatives were expressed, and this was expressed as E-selectin. and was widely used as a model for P-selectin-mediated leukocyte adhesion (Phil I ips et al., 5science250:1130.1990; Pol Ie y et al., Proc, Natl, Acad, Sci, US 88:6224.19 91; Handa et al., Biochem, Biophys, Res.

Commun, I 81: I223. 1991: Kajima et al., Bioch em, Biophys, Res, Commun, l　82　:]288.199 2)a ) (L(io cells), Newcastle disease virus (NDV) or Vibrio cholerae (VC) Cells and mAbs SNH3 and SNH3 when treated with sialidase The reactivity with 4 disappeared (Fig. 13). E-selectin-dependent HL60 adhesion is caused by N Although it only decreased by about 20-50% after treatment with DV-alidase, Vibrio or the same treated with Arthrobacter ureaphanens (AU) sialidase. Cell adhesion was reduced to about 5-10% of control values or virtually disappeared.

(NDV, VC and AU sialidase abolished SLe” expression in HL60 cells. It had the same effect on ) NDV aridase binds to the terminal Gal In contrast, Vibrio and Altrova Cacta sialidases contain terminal and internal noalic acid residues, especially α2→6-linked amino acid residues. Completely removes acid residues. These findings indicate that SLe' and SLe' are This indicates that it is not the sole epitope of lectin and P-selectin.

The effect of various mAbs on E-selectin-dependent HL60 adhesion was tested. Anti- 8Le’ mAbs SNH3 and 5NH4 are robust even under carefully controlled conditions. A HL60 cell population was generated. Therefore, HL60 by 5NH3 or 5NH4 The extent of inhibition of adhesion is determined by the separation of aggregated cells from E-selectin-coated plates. The trends were quite different.

In general, the degree of inhibition by these mAbs is similar to that of the above-mentioned Ph111 ips etc. was very small compared to the degree of suppression already described. Anti-Le’ mAb sHl (IgGz) (Singha 1st grade, Cancer Res, 5 0:1375.1990) and FN2 (1 gM) (Fukushi et al., J. Bio. l. Chem, 259:468LI984)! ;t, mAbSNH3 and 5NH The degree of inhibition was consistently higher than that of 4.

Mixtures of 5NH3 or 5NH4 with SHI or FN2 are similar to either mAb alone. The degree of inhibition was also high. The strongest inhibition occurred with a mixture of 5NH4 and SHI. (Figure 14).

If SLe' is the only effect of HL60 on E-selectin and P-selectin, If it is a pitope, anti-3Le’ mAb (e.g. 5NH3 and 5NH 4) should completely suppress selectin-dependent adhesion. HL60 cells and 5N By treatment with NDV sialidase that abolished reactivity with H3 and 5NH4 Also, E-selectin-dependent cell adhesion should be suppressed. However, H.L. Even if 60 cells were treated with NDV sialidase and then treated with 5NH3 or 5NH4, Adhesion was not further reduced. After treatment with NDV sialidase, anti-Le″mA Treatment with bsH+ or FN2 strengthens E-selectin-dependent HL60 adhesion. suppressed.

f (L60-adhesive E-selectin applied plate) on activated HUVEC in static system. A similar trend was observed for HL60 adhesion versus rate. HL60 adhesion to activated HUVECs grown in It was. Adhesion to HUVEC was previously reported by the above-mentioned Phi II ips, etc. Contrary to what was reported, the effect of anti-8Le’ mAb SNH2 or 5NH4 was extremely small. The mAb samples were 1 (Le0-HUVEC adhesive) depending on the sample. The effects on HL60 cells vary widely, with some mAbs producing robust aggregation of HL60 cells. Ta. Anti-Le’mAb SHI and FH2 Similar to the combination, -through 1 (Le0--HU　V　EC　C adhesive stronger ( SNH3 or 5NH41: in comparison). NDV sialidase is HL Although it did not significantly reduce 60-HUVEC adhesion, Vibrion aridase Reactivity almost completely disappeared.

Application of adhesion molecules or EC to glass plates in dynamic flow systems Lectin, Fibrone cutin (FN), laminin (LN), truncated E-selectin and GSL When using 10 to 50 μl of a solution with a concentration of 20 to 200 μg/ml, Board (38x75mm, Corning Gla, Corning, New York) Place it in the marked area (0.5 cm in diameter) on Dry in the refrigerator at 4°C. Soak the dry plate in PBS for 1 hour at 37°C. Then, the plate was washed thoroughly by changing PBS several times. For GSL application, add 81ml of PB. GSLjOOμg: l Restylol 200μg and Phosphatidylcholine 40 GSL-'J bosomes were prepared by adding 0 μg. GSL-liposome solution lOμm was placed on a glass plate, dried at 4 °C, and the plate was Washed with PBS in the same manner.

The amount of adsorbed molecules was determined using 125I labeling for lectin, FN or LN, and GS L-liposomes were measured using a l:'H) cholesterol label. child Under these conditions, most of the proteins, regardless of FN, LN or lectin, The entire amount was adsorbed onto the glass plate. For example, when applying 100μg/m1FN , 12.5±1°8 μg/mm' was adsorbed. Similarly, dried on a glass plate Almost all of the G5l7-liposomes were adsorbed; e.g., 200 μg/m When applying l G S L-liposome, 31.3 ± 5.2 μg GSL/ mm' was adsorbed.

Place 100 μl of suspension containing 2xlO” mouse or human EC on a glass plate. and culture in a CO2 incubator at 37'C until frontal growth is obtained. EC was applied by.

Fix the plate coated with adhesion molecules or EC in a flow chamber, and A suspension of ranoma cells was passed through the chamber as described above. B16 Cells were harvested from culture using PBS containing 0.02% EDTA and 1xl Suspended in PBS at a concentration of O'/ml.

HL60 adhesion HL60-H to activated HU VEC in dynamic flow system Effects of various mAbs and noalidases on UVEC adhesion in a dynamic flow system It was also tested. In general, the effects of mAbs are similar to those observed in static systems. It was similar to mAb SNH4 had no inhibitory effect under various shear stresses Ta. mAb S l-r I and FN2 showed moderate inhibition. Here too, 5N The most potent inhibition was obtained with the combination of H4+SHI or PH2.

Adhesion was moderately reduced by NDV noalidase and It was almost completely suppressed by turn alidase.

See Figure 15.

The above results obtained using HL60 indicate that noalic acids in carbohydrate epitopes are presence is important in conferring binding specificity for E-selectin It suggests. However, the sialic acid residue is linked to G2-3 at the terminal Gal. Alternatively, the sialic acid residue need not be located at an internal position, such as an internal Gal or can exist bound to GlcNAC. However, GIcNAc It is clear that α1→3 fuconolization is essential.

Under dynamic conditions, NDV aridase showed an inhibitory effect only at low shear stress, whereas Thus, ~lC or henoalidase significantly reduced adhesion even at high shear stress. Ta. Anti-Le”I gGmAb SHl strongly inhibits adhesion even at high shear stress. On the other hand, the effect of anti-8Le' IgG3 mAb SN) (4) was very small. Ta. The most potent inhibition was achieved by the combination of NDV noalidase + anti-Le'mAb SHI. It was born by letting it grow. The mixture of anti-L e' ten anti-8 Le' mAbs is The inhibitory effect was greater than either mAb alone.

As shown in Figure 19, at low shear stress (4 dynes/cm2), adhesion was markedly inhibited by the enzyme or mAb SNH4, whereas these The drug had no effect at high shear stress (8-16 dynes/cm'). child In contrast, VC alidase completely abolished adhesion at high shear stress. mA bsHl inhibited adhesion more strongly at low shear stress than at high shear stress, but the difference was It was relatively small.

Le' alone is clearly not sufficient as an E-selectin epitope. nothing However, an α2→3+α2→6 sialylated structure is required. Le’-liposome and and Le'-liposomes are bound to the ELAM-coated plate, and the binding by SLe' is is stronger than the binding by Le' or other glycolipids. However, these epi Topes are present on the cell surface in the form of multilayered O-glycolylated mucin-type glycoproteins. That's right. Not only S L e’ but also Le’ is the same internal G in the CHO chain. α2→6 sialylation with al or GIcNAc, or G2-6 sialylation is May exist in adjacent branched structures. Internal α1→3 fuco/ru conversion (Hakomo ri et al., Biochem, Bi.

ly+IYs, Res, Common, 113: 791, + 983 ) is a G2-6 sialylated type 2 chain structure "6-C ganglioside", It did not bind to E-selectin. Therefore, the above structure is E1. , A There is no possibility of it being an M epitope.

Example 7 Colo201 cell adhesion: Effects of various noalidase and mAbs on HL60 cells ( (expressing predominantly type 2 chain structure), in contrast to C.

1o201 cells primarily express type 1 chains and are E-selectin dependent C.

1o201 adhesion is mediated by the l-roadway epitope. Co1o20] cells in various Exposure to noalidase, treatment with various mAbs and assay for residual binding. I did it. CQ10201 and mAb CA19--9 (against SLe″I) and reactivity was almost completely suppressed by Vibrio sialidase and The extent of inhibition by vector and NDV/alidase was smaller.

In contrast, Co1o201 and mAbFH7 (SLc’ and SLe’ll ) was reduced by Arthrobacterne alidase, but The effects of Rio or NDV sialidase were minimal. Co1o20J and m AbCA3F4 (Nudelman et al., J. Biol. Chem, 261:5 487.1986) was enhanced by sialidase treatment. mAb CA]9-9 slightly suppressed Co1o201 adhesion and inhibited vibriosialidase. The effect of this was very slight (Figure 16).

The SLe” epitope present on the surface of Co1o201 cells is (])]E-Sele. Regarding cutin-dependent contact CA]9-9 is not affected, and (11) Sialida It is possible to construct the system so that it is not sensitive to the enzyme treatment. mAbFH? (N above ude, Iman, etc.) and, more precisely, the E-selectin coating pretreatment effect. The effect of mAb CA3F4 on Co1o201 adhesion to cells was enhanced by pretreatment with vibrio sialidase. Arthrobacterne Alidase promotes CoIo201 adhesion in 4. It was slightly reduced, but it did not disappear. 17th See diagram.

Col for E-selectin coated plates in a dynamic flow system.

201 adhesive In dynamic flow systems, NDV noalidase is particularly effective at high shear stress. 01 had no effect on adhesion. Anti-5Le’JmAb CA 9-9 was ineffective, whereas anti-8L e'11 mAb FH7 had a moderate inhibitory effect. It had a controlling effect. This result agrees with the one for the static system.

At both low and high shear stress, the strongest adhesion inhibition or sub-GlcNAc mAl for I-e” with α2-6 allone substitution in cA3F4 It was observed that Efficiently cleaves the terminal α2→3 noalonol bond, but the internal sialic Vibrio sialidase, which is less effective at removing acid residues, is more effective at high shear stress. reduced adhesion to some extent, but less so at low shear stress.

Similarly, mAb CA3F4 strongly inhibited adhesion at high shear stress, but at low shear stress Stress had a much smaller suppressive effect.

The combination of vibrio sialidase + mAb CA3F4 is effective at high and low shear stress. A strong inhibition occurred under both stress conditions (Fig. 18).

A similar trend was observed for NDV-alida, which specifically cleaves the terminal α2-3 sialon bond. observed for With high shear stress or low shear stress, Co1o201 adhesion , even when treated with NOV sialidase alone, mAbcA after treatment with NDV noalidase No effect was observed even when treated with 19--9. On the other hand, NDV sialidase treatment After m A b F i (7 or CA3F4 treatment, high shear stress and low shear Adhesion was strongly suppressed under both stress conditions.

Rolling speed of Co1o20] cells along E-selectin coated plate (8 m7 seconds) were measured after treatment with various sialidases and mAbs. 3 different types At shear stress, the rate is influenced by NDV alidase and mAbcA19-9. did not undergo rolling; i.e., cells that stopped (0 μm/s) started rolling again. That never happened. However, Vibrio or Arthrobacter sialidase or With mAb CA3F4, cells that had been arrested once again rolled; The rate of change depended on the shear stress. Maximum speed is CA3F4 + Vibrion Alida obtained by treatment with a combination of enzymes.

Co1o201 adhesion to activated HUVECs in a static system E-selectin application As seen for Co1o201 adhesion to cloth plates, mAbcA1 Although the effects of 9-9 and FH7 were negligible, mAb CA3F4 Strongly suppresses adhesion. ND that cleaves the terminal α2-・3 noarosyl bond ■ Even if treated with sialidase for a short time or 24 hours, adhesion does not decrease significantly. It was. When treated with vibriosialidase for 24 hours, the adhesion completely disappeared. . Short-term treatment with Vibrio and Al]. decreased significantly.

Co1o201 adhesive adhesion to activated HU VEC in dynamic flow system The inhibition was most potent with mAb CA3F4 and less effective with mAb CA19-9. It didn't work. mAblNHl or ST421 (St roud et al., J. Biol. Chem., 266:8439, +991). This resulted in significant suppression. Adhesion is not affected by NDV and annoase. However, it was strongly inhibited by vibriosialidase.

Regarding the E-selectin coated plate and I-I U V E C observed above. The effects of sialidase and mAb on Co1o201 adhesion are The type 1 chain epitope recognized by Chin is internally sialonylated and fuconlylated. This suggests that

Example 8 Truncated recombinant E L lacking transmembrane and cytoplasmic domains A M-1 can be used to fill standard chromatography columns, for example. Apply to the beads. ELAM-1 was mixed with beads at a concentration of O, 1-1 μg 7”ml. Mix and incubate the mixture to apply ELA to the bead matrix. Combine M1. A suitable incubation time is 12 to 48C to room temperature. It is 24 hours.

Wash the beads to remove unbound ELAM-1 and, if necessary, add an inert solution such as BSA. Block the carrier and wash again.

ELAM-1 coated beads can be used in a batch process or in a suitably sized color. can be filled into the tank.

Carrying carbohydrates capable of binding to ELAM-1 such as HL60 and Co1o20] cells that are known to be resistant to cancer are obtained. Cells are lysed and subjected to freeze-thaw cycles. Membrane fractions are obtained using known methods such as repeating. Membrane fractionation can be achieved by centrifugation, e.g. can get.

If the cellular source only or primarily produces carbohydrates capable of binding to ELAM-1, If the membrane sample is known to have a It is a cell source.

The membrane sample is processed using known methods to obtain a sample of membrane components, particularly cell surface carbohydrates. A fraction containing the substance is obtained. Carbohydrate-rich fractions, depending on the formant This matrix can be mixed with or passed through an ELAM-1 affinity matrix. The Trix is then washed and the carbohydrates bound to the matrix are removed, e.g. The matrix is eluted by exposure to high salt buffer.

The resulting sample comprised carbohydrates capable of binding to ELAM-1 and contained a variety of species. , TLC, HPLC, and other known techniques.

Example 9 prepared chemically using known reagents and methods (e.g., see Example 1 above); , enzymatically prepared or obtained from appropriate cells (see, e.g., Example 8 above). Expressing carbohydrates capable of binding AM-1 or carbohydrates capable of binding ELAM-1 Whole cells known to act as immunogenic antigens in suitable hosts and produce antibodies against it. Can polyclonal or monoclonal antibodies be obtained? and suitable hosts are selected based on known methods and preferences. carbohydrates, Cells, cell lysates, or membrane samples are prepared for immune responses with or without adjuvants. be administered to the host on a schedule that produces

For polyclonal antiserum, blood is drawn, serum is separated, and tested.

For monoclonal antibodies, the host animal's pancreas is removed and the cells obtained from this pancreas are lysed with appropriate myeloma cells using known techniques.

The specificity of antibodies can be determined, for example, by purification procedures using appropriate labeling reagents and reporter molecules. Tracking using an ELISA comprising engineered ELAM-1 and mAb3B7 You can

Antibodies against carbohydrates of formulas (1), (II) and (II) are used as antigens. using specific carbohydrate species that can be obtained in a screening ELISA. Ru.

In addition, the antiserum can be prepared by using cells carrying SLe'' and/or SLe', or by using cells carrying SLe'' and/or SLe'. For adsorption using a solid matrix that only binds L e″ and/or SLe″ It can be made more "monospecific". Carbohydrates that can bind to ELAM-1 The residual activity against carbohydrates of formula (1), (II) or (IIII) is due in part to It may be due to antibodies against.

Example 1O NS--1 cells were obtained from ATCC (Rockville, MD) and IO% Replenish HIFC8 RPMI 1640: Dalhecco MEM (1: I) was held at A vector containing E-selectin cDNA in vector pCDM8. Sumido (manufactured by R&D Systems, Minneapolis, Minnesota) 50μg and pSV2-ne.

(ATCC) was added to NS-1 cells (Ix107 cells) using the electro-hole method. Moved it. After 48 hours in culture, cells were injected with 650 μg/ml G418. - Gibco, Grand Island, York). .

After 15-20 days, the resulting colonies were infected with mAb (Rockville, MD). Otsuka Pharmaceutical Co., Ltd. Maryland Research Laboratory 'J-su W, N e Screening for E-selectin expression by staining with -ning. Mutants expressing high levels of E-selectin were isolated using mAbs. It was isolated by banning and limiting dilution to obtain clonality.

SLe', SLe', Le', Le', H-2, noaryl paraglobon (SPG), diallosyl I (Structure 6 in Figure 20) and dimeric SLe'' E-selectin-dependent adhesion using transfected N5-1 cells was SL'e' It is expressed as a value for adhesion to the coating plate (assumed to be 100%).

Whereas adhesion to L e’ and Le” increased slightly at high shear stress. Therefore, the absolute number of adhered cells is lower than that observed in SLe'' and SLe''. Both stress and high shear stress were much lower. The adhesion in structure l in Fig. 20 is as follows: enhanced in medium and high shear stress conditions.

The high binding capacity of structure 1 in Figure 20 can be applied to liposomes (glycoliposomes) with low concentrations of sugar. This was further clarified using lipids. SLe” Transfer to liposomes Futed N5-1 adhesion was performed by adding Le' or Ley and mixed glycoliposomes. It was enhanced when provided. The experimental results are shown in FIGS. 21 to 24.

Based on the above, although the present invention has been explained by specific embodiments, the spirit of the present invention and It will be obvious that various modifications may be made without departing from the scope.

All documents cited herein are incorporated by reference into this disclosure. be done.

Figure 2 years of life Figure 3A years of life Figure 38 years of life Figure 3C Survey months Figure 3D LacCar, ug/rnl [FN]pq/ml Inhibitor concentration (μM) Glycolipid application amount (μg) Glycolipid application amount (μg) Bond index Figure 9 Suppression % Figure 0 Figure 11A Figure 11C Figure 11D Ward sect (Ill/ x -o Ix) Heat g How much? (91,,1-1: [I4) if O9'I H (%9J-taku) grave) 091H (%94 2 doors) It's hot 709" IH Figure 15A Figure 15C Wall shear stress (dynes/cm") Wall shear stress (dynes/cm") Figure 15B 1st 5D diagram With soybean m-taku・ deception f1il illtu street Deceptive country rumors town Figure 17A Figure 17B Figure 17C Figure 18A Figure 13.8 Figure 18C (%9KugoudustU)Hokkaido09]H Fu momme 1 Figure 20 uCaI A2 Figure 20 (continued) Figure 21A Figure 21B Figure 22 @SLe”+5PG o SLe”+)j2 SLe” (μM) GSL (μM) Figure 24 Submission of translation of written amendment (Written amendment pursuant to the provisions of Article 184-8 of the Patent Law) 1. I8□198I

Claims (42)

[Claims] 1. Carbohydrates or their substituted derivatives represented by the following formula; ▲ Numerical formulas, chemical formulas, tables, etc. are available. Masu▼ [In the formula, R2 is H or α2→6-linked sialic acid, and R4 is H or α1→3-linked sialic acid. Fucose, R5 is H, α2→3-linked sialic acid, α2→3-linked NeuAcα 2→8 NeuAc disaccharide, or α2→3-linked R6-sialic acid carbohydrate (however, R 6 is one or more carbohydrates), and n is a number of 0 or more]. 2. Carbohydrates represented by the following formula or substituted derivatives thereof: ▲ Numerical formulas, chemical formulas, tables, etc. are available. Masu▼ (wherein R1 is H or α2→3-linked sialic acid, R2 is H or α2→6-linked sialic acid) sialic acid, R3 is H or α1→4-linked fucose, and n is 0 or more ). 3. Carbohydrates represented by the following formula or substituted derivatives thereof: ▲ Numerical formulas, chemical formulas, tables, etc. are available. Masu▼ (In the formula, each of R10 and R11 is galactose, Galβ1→4GlcNAc or Galβ1→3GlcNAc; R■ is galactose or Gal comprises NAC; and R9 binds lactosylceramide or carbohydrate containing oxygen groups of lipids or proteins). 4. R10 comprises a Lex epitope and R11 comprises a SLek epitope. The carbohydrate according to claim 3, comprising: 5. The carbohydrate according to claim 4, wherein R■ is Galβ1→4GlcNAc. thing. 6. Claim No. 1, wherein R9 is an oxygen group of a lipid or protein that binds a carbohydrate. Carbohydrate according to item 5. 7. R10 comprises the Le4 epitope and R11 comprises the SLe4 epitope. The carbohydrate according to claim 3, comprising: 8. The carbohydrate or substituted derivative thereof according to claim 3 represented by the following formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 9. Particularly for the carbohydrate or derivative thereof according to any one of claims 1 to 8. Antibodies that bind differently. 10. Claim 9, wherein the carbohydrate is the carbohydrate according to Claim 4. Antibodies described in Section. 11. Claim 9, wherein the carbohydrate is the carbohydrate according to claim 7. Antibodies described in Section. 12. Claim 8, wherein the carbohydrate is the carbohydrate according to Claim 8. Antibodies described in Section. 13. At least two types of charcoal involved in tumor cell or leukocyte adhesion to endothelial cells A composition comprising a hydrate. 14. Claims wherein one of the at least two types of carbohydrates is SLex. Composition according to paragraph 13. 15. 15. The composition of claim 14, further comprising Lex. 16. 15. The composition of claim 14, further comprising Ley. 17. Claim wherein said at least two carbohydrates include Le4 and SLe■. A composition according to scope item 13. 18. 14. The composition of claim 13, further comprising liposomes. 19. A composition comprising at least two types of antibodies, wherein each of the two types of antibodies each of which is the composition according to any one of claims 13 to 17. A composition that specifically binds to one of at least two carbohydrates. 20. 20. The composition of claim 19, wherein the antibody specifically binds to SLex. 21. Claims further comprising an antibody that specifically binds to Lex or Ley Composition according to paragraph 20. 22. 20. The composition of claim 19, wherein the antibody specifically binds to SLea. 23. Claims further comprising an antibody that specifically binds to Lea or Leb Composition according to paragraph 22. 24. at the end ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ Cell-cell interactions mediated by ELAM-1 in cells expressing a type 1 chain comprising A method of inhibiting Le4, the method comprising: exposing said cell to an antibody that specifically binds to Le4. A method that includes steps. 25. 25. The method according to claim 24, wherein the antibody is CA3FA or FH7. . 26. A method of inhibiting ELAM-1-mediated cell-cell interactions between cells, the method comprising: at least one antibody that specifically binds to a carbohydrate capable of binding to AM-1; A method comprising the step of exposing cells. 27. The carbohydrates capable of binding to ELAM-1 include SLe', hybrid Le x/SLex or hybrid Lea/SLea according to claim 26. How to put it on. 28. Claim 27, wherein the carbohydrate has a structure represented by the following formula: Method described. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 29. Claim 26, comprising an antibody that specifically binds to SLex. the method of. 30. Claim 29 further comprising an antibody that specifically binds to Lex. Method described. 31. Claim 26, comprising an antibody that specifically binds to SLea. the method of. 32. Claim 31 further comprising an antibody that specifically binds to Lea. Method described. 33. comprising an antibody that specifically binds to carbohydrates that do not bind to ELAM-1 A method according to claim 26. 34. The carbohydrate that does not bind to ELAM-1 is Lex, Leγ, Lea or 34. The method of claim 33, wherein the Leb. 35. The antibody that specifically binds to SLex is SNH3 or SNH4, and L Claim 30, wherein the antibody that specifically binds to ex is SH1 or FH2. The method described in. 36. A method of inhibiting ELAM-1-mediated cell-cell interactions between cells, the method comprising: AM-1, carbohydrates that can bind to ELAM-1, sialidase, ELAM-1 Antibodies that specifically bind and carbohydrates that can bind to ELAM-1. exposing said cells to at least one selected from the group consisting of antibodies. How to do it. 37. The carbohydrate capable of binding to ELAM does not contain α2→3-linked sialic acid. 37. The method of claim 36. 38. Claim 36, wherein said carbohydrate capable of binding to ELAM is Lex. The method described in. 39. The carbohydrate capable of binding to ELAM does not contain α2→6-linked sialic acid. 37. The method of claim 36. 40. The antibody that specifically binds to carbohydrates capable of binding to ELAM is α2→3 Antibodies that specifically bind to carbohydrates that can bind to ELAMs containing bound sialic acids. 37. The method of claim 36. 41. The antibody that specifically binds to carbohydrates capable of binding to ELAM is α2→6 Antibodies that specifically bind to carbohydrates that can bind to ELAMs containing bound sialic acids. 37. The method of claim 36. 42. The antibody that specifically binds to carbohydrates capable of binding to ELAM binds to Lex. 37. The method of claim 36, which is a specifically binding antibody.