WO1992004048A2

WO1992004048A2 - Inhibition of metastasis potential and invasiveness by oligosaccharides or oligosaccharide antigens or antibodies

Info

Publication number: WO1992004048A2
Application number: PCT/US1991/006202
Authority: WO
Inventors: Tatsushi Toyokuni; Sen-Itiroh Hakomori
Original assignee: The Biomembrane Institute
Priority date: 1990-08-30
Filing date: 1991-08-29
Publication date: 1992-03-19
Also published as: WO1992004048A3; EP0546122A1; AU8667791A; AU659808B2; CA2089375A1; JPH06501008A

Abstract

Agents and methods for inhibiting metastasis potential of tumor cells are provided. Metastasis potential of tumor cells may be inhibited by an agent selected from the group consisting of (a) tumor-associated carbohydrate antigens (TACAs) that exhibit differential prognostic significance, (b) antibodies that specifically bind to these antigens, (c) oligosaccharide components of these antigens, and (d) conjugates of these antigens or oligosaccharides, wherein the agent inhibits the metastasis potential of the preparation. Additional oligosaccharides for use within the methods and compositions provided herein include lactose and lactose derivatives. The present invention also discloses conjugates comprising oligosaccharides and poly(ethylene glycol).

Description

INHIBITION OF METASTASIS POTENTIAL AND INVASIVENES BY OLIGOSACCHARIDES OR OLIGOSACCHARIDE ANTIGENS OR ANTIBODIES

Technical Field

The present invention is generally directed toward the inhibition of tumor cell metastases and invasiveness, and more specifically, toward such inhibition through the use of agents including tumor-associated carbohydrate antigens and their oligosaccharide derivatives.

Background of the Invention

Despite enormous investments of financial and human resources, cancer remains one of the major causes of death. Current cancer therapies cure only about fifty percent of the patients who develop a malignant tumor. In most human malignancies, metastasis is the major cause of death.

Metastasis is the formation of a secondary tumor colony at a distant site. It is a multistep process of which tumor invasion is the first step. Tumor cells locally invade host tissue barriers, such as the epithelial basement membrane, to reach the interstitial stroma, where they gain access to blood vessels (or lymphatic channels) for further dissemination. After invading the endothelial layer of the vessel wall, the circulating tumor cells are dislodged into the circulation and arrest in the precapillary venules of the target organ by adherence to endothelial cell lumenal surfaces, or exposed basement membranes. The tumor cells again invade the vascular wall to enter the organ parenchyma. Finally, the extravasated tumor cell grows in a tissue different from where it originated.

In most human malignancies, distant metastases are often too small to be detected at the time the primary tumor is treated. Furthermore, widespread initiation of metastatic colonies usually occurs before clinical symptoms of metastatic disease are evident. The size and age variation in metastases, their dispersed anatomical location, and their heterogeneous composition are all factors that hinder surgical removal and limit the concentration of anticancer drugs that can be delivered to the metastatic colonies.

Due to the difficulties in the current approaches to the treatment and prevention of metastases, there is a need in the art for improved methods and compositions for inhibiting metastasis potential of tumor cells. The present invention fills this need, and further provides other related advantages.

Summary of the Invention

Briefly stated, the present invention provides a variety of agents and methods for inhibiting metastasis potential and invasiveness of tumor cells. In one aspect of the present invention, a method for inhibiting tumor cell metastasis potential within a biological preparation is provided. The method comprises incubating the biological preparation with at least one agent selected from the group consisting of (a) tumor-associated carbohydrate antigens that exhibit differential prognostic significance, (b) antibodies that specifically bind to these antigens, (c) oligosaccharide components of these antigens, and (d) conjugates of these antigens or oligosaccharides, the agent inhibiting the metastasis potential of the preparation. Suitable biological preparations include cell cultures and biological fluids.

Another aspect of the present invention provides agents for use within the manufacture of a medicament for inhibiting metastasis potential of tumor cells in a warm-blooded animal. An agent is selected from the group consisting of (a) tumor-associated carbohydrate antigens that exhibit differential prognostic significance, (b) antibodies that specifically bind to these antigens, (c) oligosaccharide components of these antigens, and (d) conjugates of these antigens or oligosaccharides, wherein the agent is capable of inhibiting tumor cell metastasis potential.

Within a related aspect, the present invention provides a variety of glycoconjugates useful for prolonging the in vivo lifetime of oligosaccharides. The conjugates comprise an oligosaccharide coupled to poly(ethylene glycol).

Additional oligosaccharides for use within the present invention include lactose, lacto-N-tetrose, methyl β-D -lactoside and phenyl β-D- thiolactoside. Oligosaccharides may be used individually or in combination with one another.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. Brief Description of the Drawings

Figure 1 graphically illustrates survival of cancer patients with or without expression of a defined tumor associated carbohydrate antigen (TACA) in their tumors. Panel A represents the expression of H/Le^y/Le^b antigen in lung squamous cell carcinoma as determined by MAb MIA-15-5. Panel B represents sialosyl-Le^x expression in colonic cancer. Pane! C represents sialosyl-Tn expression in colonic cancer. Panel D represents sialosyl-Tn level in sera of ovarian cancer patients.

Figure 2 graphically illustrates the effects of methyl β -D-lactoside or phenyl β-D-thiolactoside on the number and size of lung colony deposits of BL6 cells. BL6 were preincubated with control medium, 0.1 M methyl β- D-lactoside ("Me-β-lactoside"), or 0.1 M phenyl β-D-thiolactoside ("phe-β-S-lactoside). 2 x 10⁴ cells were injected intravenously into C57/BL mice. Lung colony numbers were counted at 21 days, and colonies were classified on the basis of diameter (> 1 mm vs. < 1 mm), as indicated for each column. Colony numbers are expressed per single lung. Number of experiments ("n") is indicated in parentheses.

Figure 3 graphically illustrates the effect of prior administration of methyl β-D-lactoside on the number and size of lung colony deposits of BL6 cells. Methyl β-D-lactoside (1 ml dosage) was injected intraperitoneally into C57/BL mice. After 10 minutes, B16 melanoma cells were injected intravenously. Lung colonies were counted and sized at 19 days. Group A represents control animals (not administered methyl β-D-lactoside) and groups B and C represent animals injected with 0.25 M and 0.5 M methyl β-D-lactoside, respectively. For each group, column 1 represents the total number of colonies, column 2 the number of colonies with diameter > 1 mm, and column 3 the number of colonies with diameter < 1 mm. Number of experiments is expressed as "n".

Figure 4 graphically illustrates the metastasis-inhibitory effect of methyl(Me)-β-lactoside. Tumor cells were intravenously injected, followed by intraperitoneal injection of: PBS control (A); 0.25 M Me-β-lactoside (B); 0.5 M Me-β-lactoside (C); 0.5 M lactose (D); 0.25 M N-acetyllactosamine (E); or 0.5 M Me-β-galactoside (E).

Figure 5 graphically illustrates that melanoma cell adhesion on LacCer is based on GM3-LacCer interaction. The order of metastatic potential is BL6>F10>F1 > >WA4. Panel A shows the order of melanoma cell adhesion on LacCer-coated solid phase. Panel B shows the order of melanoma cell adhesion on LacCer/Fibronectin (FN) co-coated solid phase. Panel C shows integrindependent adhesion.

Figure 6 graphically illustrates the melanoma cell (BL6) adhesion on LacCer (Panel A) and on endothelial cells (HUVEC) (Panel B) are inhibited by LacCer and GM3. Figure 7 graphically illustrates H-Le^y and H-H interaction. Panel A shows H₁-liposome binding to various glycolipids. Panel B shows Le^y-liposome binding to various glycolipids. Detailed Description of the Invention

As noted above, the present invention is directed towards agents and methods for the inhibition of tumor cell metastasis potential and invasiveness. Numerous tumor cells possess the ability to metastasize, i.e., to form a secondary tumor colony at a distant site. Sources of malignant tumor cells include melanoma, lung, breast, colorectal and urogenital cancers, such as bladder and prostate cancers. Within the present invention, the metastasis potential of tumor cells may be inhibited (i.e., inhibiting the ability of tumor cells to metastasize) through the use of (a) tumor-associated carbohydrate antigens (TACAs); (b) antibodies directed to these TACAs; (c) oligosaccharide components of these TACAs; or (d) conjugates of such TACAs, such as multivalent conjugates of lysyllysine or TACA-bearing glycosphingolipid (GSL) liposomes.

TACA epitopes play essential roles in tumor cell adhesion through their interaction with endothelial cells, platelets and basement membranes, whereby tumor metastasis and invasion may occur. The mechanism of adhesion may be based upon carbohydrate (CHO) - CHO interaction, CHO-lectin interaction or selectin family interaction. Adhesion of various tumor cells on activated endothelial cells and platelets is mediated primarily by the Leccam or selectin superfamily (e.g., ELAM-1, GMP-140). Colo205 tumor cells, which express type 1 chain sialosyl-Le^a (SA-Le^a) but not sialosyl-Le^x (SA-Le^x), adhere to endothelial cells. This adhesion was inhibited by anti-SA-Le^a MAb, but not by SA-Le^x MAb. These findings suggest that not only SA-Le^x, but also SA-Le^a, are the important ligands recognized by ELAM-1 and GMP-140.

Within the present invention, tumor metastasis and invasion is inhibited by blocking tumor cell adhesion, thereby significantly reducing or eliminating the spread of metastatic cells. TACAs suitable for use within the present invention are those showing differential prognostic significance (i.e., TACAs that may be clearly correlated with invasive or metastatic potential). Within the context of the present invention, such TACAs may be distinguished through a comparison of invasiveness, metastasis and clinical prognosis of similar tumors showing expression vs. non-expression of such TACAs. Preferred TACAs for use within the present invention include H/Le^y/Le^b, sialosyl-Le^x (SA-Le^x), sialosyl-Le^a (SA-Le^a), and sialosyl-Tn (SA-Tn). Derivatives of such TACAs include dimeric Le^x, sialosyl-dimeric Le^x and trifuscosyl Le^x .

The structures of sialosyl-Le^x (structure 1), sialosyl-dimeric Le^x (structure 2), dimeric Le^x (Structure 3), trifucosyl L e^x (structure 4), Le^b (structure 5), H (structure 6), SA-Le^a (structure 7), SA-Tn (structure 8), and GM3 (structure 9) are shown below.

Structure 1:

NeuAcα2→3Galβ1→4GlcNAcβ1-3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→R F

Structure 2:

NeuAcα2-3Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→R

Structure 3:

Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→R

Structure 4:

Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→R

Structure 5:

Fucα1-2Galβ1→3GlcNAcβ1→3Galβ1→R

Structure 6:

Fucα1→2Galβ1→3GlcNAcβ1-3Galβ1→R Structure 7:

NeuAcα2→3Galβ1→3GlcNAcβ1→3Galβ1→R

Structure 8:

NeuAcα2→6GalNAcα1→O-Ser/Thr

Structure 9:

NeuAcα2→3Galβ1→4Glcβ1→Cer

As noted above, TACAs for use within the present invention exhibit a differential prognostic significance. By way of example, such a differential prognostic significance may be illustrated by the fact that tumors expressing H/Le^y/Le^b antigens (as defined by MAb MIA-15-5) showed much worse patient prognosis than tumors not expressing these antigens. For instance, as shown in Figure 1 A, patients with squamous cell lung carcinoma expressing H/Le^y/Le^b had only an 11% survival over a 5-year period (i.e., 89% died), whereas comparable patients not expressing H/Ley/Le" had an approximately 62% survival over this period. Similar results were obtained for tumors showing expression vs. non- expression of SA-Le^x and SA-Tn antigens. More specifically, as shown in Figure 1B, patients with colonic cancer expressing SA-Le^x had only a 15% survival over a 5-year period, whereas comparable patients not expressing this antigen had an approximately 50% survival over this period. In a separate study, the 5-year survival of patients with early-stage colonic cancer not expressing SA-Tn was 100%, as compared to 75% for patients who expressed SA-Tn (see Figure 1C). As shown in Figure 1D, similar but more obvious differences were observed in patients with ovarian cancer showing expression vs. non-expression of SA-Tn antigen.

As noted above, antibodies to suitable TACAs may also be employed within the context of the present invention. As used herein, such antibodies include both monoclonal and polyclonal antibodies and may be intact molecules, a fragment of such a molecule, or a functional equivalent thereof. The antibody may be genetically engineered. Examples of antibody fragments include F(ab')₂, Fab', Fab and Fv. Briefly, polyclonal antibodies may be produced by immunization of an animal and subsequent collection of its sera. Immunization is accomplished, for example, by a systemic administration, such as by subcutaneous, intraspienic or intramuscular injection, into a rabbit, rat or mouse. It is generally preferred to follow the initial immunization with one or more booster immunizations prior to sera collection. Such methodology is well known and described in a number of references.

While polyclonal antibodies may be employed in the present invention, monoclonal antibodies (MAbs) are preferred. MAbs suitable within the present invention include those of murine or human origin, or chimeric antibodies such as those which combine portions of both human and murine antibodies (i.e., antigen binding region of murine antibody plus constant regions of human antibody). Human and chimeric antibodies may be produced using methods known by those skilled in the art Human antibodies and chimeric human-mouse antibodies are advantageous because they are less likely than murine antibodies to cause the production of anti-antibodies when administered clinically.

MAbs may be generally produced by the method of Kohler and Milstein (Nature 256:495-497, 1975; Eur. J. Immunol. 6:511-519, 1976), as well as by various techniques which modify their initial method (see Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is herein incorporated by reference in its entirety). Briefly, the lymph nodes and/or spleens of an animal immunized with one of the TACAs or their oligosaccharide components are fused with myeloma cells to form hybrid cell lines ("hybridomas" or "clones"). Each hybridoma secretes a single type of immunoglobulin and, like the myeloma cells, has the potential for indefinite cell division. It may be desirable to couple such molecules to a carrier to increase their immunogenicity. Suitable carriers include keyhole limpet hemocyanin, thyroglobulin, bovine serum albumin and derivatives thereof. An alternative to the production of MAbs via hybridomas is the creation of MAb expression libraries using bacteriophage and bacteria (e.g., Sastry et al., Proc. Natl. Acad. Sci USA 86:5728, 1989; Huse et al., Science 246:1275, 1989). Selection of antibodies exhibiting appropriate specificity may be performed in a variety of ways which will be evident to those skilled in the art

Representative examples of MAbs suitable for use within the present invention include MlA-15-5 (Miyake and Hakomori, Biochem. 30:3328, 1991), as well as the MAbs cited within Hakomori, Advances In Cancer Research 52:2 57-331, 1989.

As discussed above, oligosaccharide components of suitable TACAs may also be used within the present invention. As used herein, the term "oligosaccharides" includes naturally derived oligosaccharides, synthetically prepared, and derivatives of either, including portions of a TACA oligosaccharide component

Additional oligosaccharides useful within the present invention include lactose and lactose derivatives, such as methyl β-D -lactoside, lacto-N- tetrose (Galβ1→3GlcNAcβ1→3Galβ1→4Glc), and phenyl β-D-thiolactoside. Other lactose derivatives may also be used, including ethyl or phenyl lactoside and methyl or ethyl thiolactoside.

Other oligosaccharides suitable for inhibiting metastasis potential of cells of a particular tumor may be identified based upon determination of the structure of specific carbohydrate chain(s) which are involved in the tumor's ability to metastasize. The identification of carbohydrate-containing molecules involved in a tumor's ability to metastasize may be accomplished in a variety of ways, including through the use of glycosidases and inhibitors of glycosyltransferases. The structure of carbohydrates bound to either lipids or proteins may be determined based on degradation, mass spectrometry, including electron-impact direct-probe (El) and fast atom bombardment (FAB), and methylation analysis (techniques described, for example, in Nudelman et al., J. Biol. Chem. 261:5487-5495. 1986). Degradation analysis may be accomplished chemically and/or enzymatically, e.g., by glycosidases. The carbohydrate sequence suggested by degradation analysis may be determined by methylation analysis (Hakomori, J. Biochem. 55:205-208, 1964) followed by chemical ionization mass spectrometry of permethylated sugars (Stellner et al., Arch. Biochem. Biophys. 155:464-472, 1974; Levery et al., Meth. Enzymol. 138:13-25, 1987). Alternatively, or in conjunction with these techniques, El mass spectrometry may be performed on permethylated glycans or after the appropriate degradation of intact glycans (Kannagi et al., J. Biol. Chem. 259:8444-8451, 1984; Nudelman et al., J. Biol. Chem. 263:13942-13951, 1988). Homogeneity of the carbohydrate sequence may be demonstrated based on various chemical and physical criteria, including proton NMR spectroscopy of intact or methylated glycans and FAB mass spectrometry. Once the carbohydrate sequence has been determined, it will be evident to those of ordinary skill in the art to select an appropriate oligosaccharide for inhibiting the tumor cell's metastasis potential. As briefly discussed above, conjugates of suitable TACAs or oligosaccharide components thereof, such as multivalent conjugates with lysyilysine or TACA-bearing giycosphingolipid (GSL) liposomes, may also be used within the present invention.

The components of the conjugate may be covalently coupled to one another either directly or via a linker group. A direct reaction between components is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one component may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acyl halide, or with an alkyl group containing a good leaving group, e.g., a halide, on the other.

It may be desirable to covalently couple components via a linker group. It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the Pierce Chemical Co. catalog, Rockford, IL), may be employed as the linker group. A linker group can serve to increase the chemical reactivity of a substituent and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of functional groups on components which would not otherwise be possible. For example, a carboxyl group may be activated. Activation of a carboxyl group includes formation of an "active ester," such as a succinimidyl ester. The term "active ester" is known to refer to esters which are highly reactive in nucleophilic substitution reactions.

Alternatively, it may be desirable to produce conjugates in which the components are non-covalently linked. For example, one or more TACAs may be incorporated into the outer surface of GSL Iiposomes in a similar manner as previously described (Eggens et al., J. Biol. Chem. 264:9476-9484. 1989; Batzri and Korn, Biochim. Biophys. Acta 298:1015-1019, 1973; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA 75:4194-4198. 1978).

It may be desirable to increase the in vivo lifetime of an oligosaccharide. As disclosed within the present invention, oligosaccharides may be coupled to (i.e., covalently bonded to) a straight-chain amphiphilic polymer, such as poly(ethylene glycol). A representative example of a method for producing an oligosaccharide-poly(ethylene glycol) conjugate is the reaction of an oligosaccharide, which has been derivatized to contain a succinimidyl group, with a poly(ethylene glycol) having a terminal amino group. The latter compound has a general formula of NH₂-(CH₂CH₂-O)_n-CH₃, where n typically averages 44.7 (i.e., molecular weight of about 2,000) to 112.9 (i.e., molecular weight of about 5,000).

The inhibition of metastasis potential of tumor cells has a variety of in vitro and in vivo uses, e.g., treatment of isolated tumor cells or tumor-bearing hosts. Regarding in vitro aspects, as noted above, the present invention provides a method for inhibiting tumor cell metastasis potential within a biological preparation. The method comprises incubating a biological preparation with at least one agent selected from the group consisting of (a) tumor-associated carbohydrate antigens that exhibit differential prognostic significance, (b) antibodies that specifically bind to these antigens, (c) oligosaccharide components of these antigens, and (d) conjugates of these antigens or oligosaccharides, the agent inhibiting the metastasis potential of the preparation. Suitable biological preparations include cell cultures and cell suspensions in biological fluids, such as blood, urine, lymph, synovial and cerebrospinal fluid. TACAs, oligosaccharides or conjugates thereof will generally be incubated at a final concentration of about 0.1 to 1 M, and typically at about 02 to 03 M. Incubation is typically performed for 5 to 15 minutes at 37°C. After treatment of a biological preparation, the preparation may be injected or implanted in an animal, e.g., to confirm effectiveness of the inhibition of metastasis potential.

The present invention also provides uses of the agents in medicaments and methods for inhibiting tumor cell metastasis potential in a warm-blooded animal, such as a human. One or more agents is selected from the group consisting of (a) tumor-associated carbohydrate antigens that exhibit differential prognostic significance, (b) antibodies that specifically bind to these antigens, (c) oligosaccharide components of these antigens, and (d) conjugates of these antigens or oligosaccharides, the agent inhibiting the metastasis potential of the preparation. TACAs, oligosaccharides or conjugates thereof will generally be administered at a concentration of about 0.1 to 1 M and typically at about 0.2 to 03 M. It will be evident to those skilled in the art how to determine the optimal effective dose for a particular substance, e.g., based upon in vitro and in vivo studies in non-human animals. A variety of routes of administration may be used. Typically, administration will be intravenous or intracavitoiy, e.g., in pleural or peritoneal cavities, or in the bed of a resected tumor. A TACA, Ab, oligosaccharide or derivative as discussed above may be administered in combination with a pharmaceutically acceptable carrier or diluent, such as physiological saline. Moreover, such substances may be administered in combination with an immunotherapeutic or chemotherapeutic agent When a combination of such a substance and an agent is desired, each compound may be administered sequentially, simultaneously, or combined and administered as a single composition. Diagnostic techniques, such as CAT scans, may be performed prior to and subsequent to administration to confirm the effectiveness of the inhibition of metastatic potential.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1

SYNTHESIS OF 1-ACΓOSE DERIVATIVES

A. Methyl β-D-lactoside

Heptaacetyllactosylimidate (Zimmeπnann et al., J. Carbohydr. Chem. 7:435, 1988) was reacted with methanol in dry dichloromethane containing trimethylsilyl trifluoromethanesulfonate according to standard procedure (Grundler and Schmit, Liebigs Ann. Chem. 1984:1826, 1984). Purification by silica-gel column chromatography (toluene/EtOAc 1:1), followed by de-O-acetylation with 0.01 M sodium mefhoxide, gave methyl β-D-lactoside in 68% yield from the imidate: m.p. 211-212°C (lit. 205°C, Smith and van Cleve, J. Am. Chem. Soc. 77:3159, 1955); [α]_D + 13° (c 6.9, H₂O) (lit + 1°, c 5.0, H₂O), ibid).

B. Phenyl β-D-thiolactoside

Lactose octaacetate (Hudson and Kunz, J. Am. Chem. Soc. 47:2052, 1926) was treated with thiophenol and SnCl₄ (Nicolaou et al., J. Am. Chem. Soc. 110:7910. 1988) in dichloromethane at 0°C to give phenyl heptaacetyl β-D-thiolactoside in 80% yield. This product was deacetylated with NaOMe in MeOH and neutralized with Amberlyst^® 15. Purification of the product on a BioGel^® P-2 column using water as an eluent, followed by lyophilization of the sugar-containing fraction, left phenyl β-D-thiolactoside as a white amorphous powder.

C. Lacto-N-tetrose

This oligosaccharide (Galβ1→3GlcNAcβ1→3Galβ1-4Glc) was prepared from human milk by pretreatment with ethanol and recycling BioGel P-2 column chromatography with water as eluent, followed by reversed-phase (C₁₈) high pressure liquid chromatography with water (Dua and Bush, Anal. Biochem. 133:1. 1983). The ¹H-NMR spectrum was superimposed on that of the authentic sample (BioCarb Chemicals, Lund, Sweden). D. Poly(ethylene glycol) derivative of β-D-lactoside The reaction scheme is as follows:

The poly(ethylene glycol) derivative of β-D-lactoside was prepared from readily available 3-succinimidooxycarbonylpropyl O-(2, 3, 4, 6-tetra-O-acetyl- β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (1) and poly(ethylene glycol) methyl ether (average M.W.2000; Aldrich Chemical, Milwaukee, WI) having terminal amino group (2) (Zalipsky et al., Eur. Polym. J. 19:1177, 1983). Treatment of 1 (100 mg, 0.12 mmol) and 2 (163 mg, 0.082 mmol) in dry N,N-dimethylformamide (2 mL) at room temperature for 2 hours gave, after chromatography on LH-20 with acetone as an eluent the β-D-lactoside heptaacetate 3 in 91% yield: [α]_D -53° (c 0.5, chloroform). A subsequent saponification of 3 with 0.05 M sodium hydroxide at room temperature for one hour, followed by lyophilization, afforded the desired lactoside 4 quantitatively: [α]_D -2.4° (c 1.0, chloroform).

Example 2

EFFECT OF LACTOSE AND LACTOSE DERIVATIVES ON METASTATIC POTENTIAL OF B16

MELANOMA CELLS

A. Cells and Animals

The highly metastatic BL6 clone of B16 melanoma cell line was obtained originally from Dr. Jean Starkey (Montana State Univ., Bozeman, MT), and clones were reselected in syngeneic C57/BL mice according to their metastatic potential. C57/BL mice were maintained in plastic cages under filtered air atmosphere and provided with water and food pellets ad lib. Cells were cultured in RPMI 1640 supplemented with 2 mM glutamine and 10% fetal calf serum (FCS), and detached with phosphate buffered saline (PBS) containing 2 mM EDTA. Viability was tested by trypan blue exclusion test

B. Effects of Oligosaccharides on Metastatic Potential

A suspension of BL6 cells (1-3 x 10⁶ cells/ml RPMI 1640 medium) was prepared and aliquots were incubated in the presence or absence of various oligosaccharides at various concentrations, at 37°C for 5-10 minutes. Following incubation, typically, 3 x 10⁴ or 2 x 10⁴ cells (with or without oligosaccharide pretreatment) per 200 μl were injected via tail vein into 8-week-old female mice. After 18-21 days, mice were killed, lungs were fixed in 10% formaldehyde in PBS (pH 7.4), and tumor cell colonies were counted under a dissecting microscope, thus providing background values of metastatic melanoma colony number in lung under these conditions. Data on number and size of colonies were statistically treated by the analysis of variance (ANOVA) procedure. Colonies with a diameter of 1 mm or greater were considered large-size and those with a diameter less than 1 mm were considered small-size.

For one experiment, BL6 cells were incubated with various concentrations of lactose, lacto-N-tetrose (Galβ1→3GlcNAcβ1→3Galβ1→4Glc), methyl β-D-lactoside, or phenyl β-D-thiolactoside for various durations. In the majority of experiments, a concentration of 0.1 M was used and cells were incubated at 37°C for 10 minutes, separated from sugar-containing medium by mild centrifugation at 400 x g for 10 minutes, resuspended in RPMI 1640, and injected (3 x 10⁴ cells in 02 ml suspension) via tail vein. For some experiments, 2 x 10⁴ cells were injected and colonies were counted at 21 days. Viability and cell growth ability of BL6 cells after incubation in various sugar solutions were tested by trypan blue exclusion test, and by plating in RPMI 1640 culture under normal conditions in vitro, as well as by subcutaneous inoculation in age-matched C57/BL mice in order to test tumor growth.

Lactose and lacto-N-tetrose showed 26% and 36% reductions, respectively, of metastatic colomes in lung when BL6 cells were preincubated with these sugars followed by intravenous injection of cells under identical conditions. Treatment of BL6 cells with 0.1 M, 0.01 M, or 0.005 M methyl β-D-lactoside under the same conditions as above resulted in (respectively) a 43%, 16%, and 8% reduction of metastatic lung colony number compared to control. The significant reduction caused by 0.1 M methyl β-D-lactoside was reproduced in three separate experiments and the reduction was found to be consistently between 35% and 45%.

In a second, completely independent series of experiments, treatment with methyl β-D-lactoside or phenyl β-D-thiolactoside under different conditions also produced a significant reduction of metastatic colonization, i.e., total colony number was reduced to 35% or 50% of control value following preincubation with methyl β-D-lactoside or phenyl β-D-thiolactoside re spectively. Reduction of larger-size colomes was more apparent than that of smaller colomes in all experiments, particularly those with phenyl β-D-thiolactoside (Figure 2). Methyl β -D-lactoside and phenyl β-D-thiolactoskc both showed a slight in vitro stimulatory effect on cell number increase and on thymidine incorporation. Thus, the inhibitory effect on tumor deposition is not related to the effect on cell growth in vitro or in vivo.

In a separate experiment, the effect of methyl β-D-lactoside on melanoma cell metastasis was determined after administration of the oligosaccharide, followed by inoculation with tumor cells. Specifically, a one ml dosage of methyl β-D-lactoside (at a concentration of 0.25 M or 03 M) was injected intraperitoneaily in mice. After 10 minutes, B16 melanoma cells were injected intravenously. Lung colonies were counted 19 days later. Injection of methyl β-D-lactoside in advance of inoculation with tumor cells resulted in a significant reduction of lung metastatic colony formation (Figure 3).

In addition, the observations on the metastasis-inhibitory effect of methyl-β-lactoside have been extended to separate methyl-β-lactoside injection; i.e., tumor cells were intravenously injected, followed by intraperitoneal injection of methyl- β-lactoside. In these experiments, injection of 0.25-03 M methyl-β- lactoside reduced lung metastatic colony number by 40%-70% (see Figure 4; A=PBS control, B=0.25 M Me-β-lactoside; C=0.5 M Me-β-lactoside; D=0.5 M lactose; E=0.25 M N-acetyllactosamine; F=0.5 M Me-β-galactoside; intraperitoneal injection).

In a separate experiment, mouse melanoma B16 variants showing different degrees of metastatic potential (BL6/F10/F1/WA4) showed the same order of expression of GM3 ganglioside, which was previously identified as a melanoma-associated antigen (Hirabayashi et al., J. Biol. Chem. 260:13328. 1985; Nores et al., J. Immunol. 122:3171, 1987). GM3 interacts with LacCer, which is highly expressed on endothelial cells. The order of adhesion of the B16 variants onto LacCer-coated solid phase or onto endothelial cells was also in the same order as their metastatic potential. In contrast, integrin-dependent adhesion of the B16 variants was approximately equal for BL6, F10, and F1 (see Figure 5). These observations suggest that B16 adhesion of LacCer is based on molecular GM3-LacCer interaction. It has also been demonstrated that B16 melanoma adhesion on endothelial cells is inhibited not only by methyl-β-lactoside but also by LacCer liposome, Gg3Cer liposome, and GM3 liposome (see Figure 6).

Example 3

EXPRESSION OF SIALOSYL-DIMERIC LE^X ON HUMAN LUNG ADENOCARONOMA CELL LINES AND

METASTATIC POTENTIAL

A. Cell lines

KUM-LK-2 is a human non-adenocarcinoma cell line characterized as producing spontaneous lung metastasis in nude mice. After screening 35 human carcinoma cell lines grown in nude mice, only this cell line produced metastatic deposits in nude mouse lung. KUM-LK-2 was used as the parent cell line to obtain, by limiting dilution technique, sub-cell lines producing lung metastasis upon IV injection.

The procedure for the limiting dilution technique was as follows. KUM-LK-2 was cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% FCS (Hyclone, Logan, UT) at 37°C in a 5% CO₂/95% air atmosphere. Cells were treated briefly with 2 mM EDTA solution and washed twice with RPMI 1640 to make a single cell suspension in RPMI with 10% FCS. Cell viability was > 98% as determined by trypan blue exclusion staining. A cell suspension containing 1 cell per 100 μl was transferred to each well of a 96-well microtiter plate (Corning Glass Works, Corning, NY) and cultured continuously for 24 hours. Each well was then examined by phase contrast microscopy.

Three cell lines (HAL-8, HAL-24, and HAL-33) with different metastatic potential ("MP") were selected out of 25 clones obtained by limiting dilution technique on the basis of stable cell morphology. These 25 clones were originally selected from 63 clones showing stable morphology as well as consistent in vitro cell growth. All of these clones produced spontaneous lung metastasis. However, upon IV injection, clear differences were observed among the clones in terms of lung metastatic deposit formation. Two clones with high MP, five with low MP, and 18 with no MP were distinguished. Through repeated selection by IV injection of these clones, the most stable sub-cell lines showing consistent MP were established. These were HAL-8, -33, and -24, showing high, low, and no MP, respectively, to nu/nu mouse lung (see Table 1 below). Judging by macroscopic and microscopic examination, none of these three sub-cell lines showed metastasis in other organs or lymph nodes. The sub-cell lines represent stable variants originally present in KUM-LK-2. Based on chromosome analysis, these subclones are independent.

Nude mice were injected (2 x 10⁵ cells) in the tail vein at various generation times as indicated. 56 days after injection, mice were killed and metastatic nodules on lung surface were counted under dissecting microscope. b Mean of 6 animals (range in parentheses). B. Expression of Cell Surface Carbohydrate Epitopes

The cell surface expression of various carbohydrate epitopes was analyzed by cytofluorometry using various monoclonal antibodies (MAbs) directed to Le^x (MAb SH1), sialosyl-Le^x (MAb SH4), sialosyl-dimeric Le^x (MAb FH6), T (MAb HH8), Tn (MAb 1E3), and sialosyl-Tn (MAb TKH2). All antibodies used were culture supematants from their respective hybridomas, adjusted as 10 μg/ml of immunoglobulin.

Cells were detached from culture flask with 0.25% trypsin, 2 mM EDTA solution; 1 x 10⁵ cells were prepared for each MAb treatment Cells were incubated with a MAb for 1 hour at 4°C and washed 2 times with RPMI 1640. Goat anti-mouse IgG or IgM-FTTC (Boehringer-Mannheim, Indianapolis, IN), diluted 50 times with PBS, was then added and incubated 30 minutes at 4°C. Finally, cells were washed 3 times, resuspended with PBS, and applied to an EPICS PROFILE flow cytometiy (Epics, Hialeah, FL). These experiments were repeated with three different cell generations.

Patterns of expression of six carbohydrate epitopes (defined by their respective MAbs) on sub-cell lines HAL-8, -24, and -33 showed nearly identical profiles (as did the protein profiles for the three sub-cell lines), except in the case of sialosyl-dimeric Le^x. In particular, HAL-8, -24, and -33 were found to highly and equally express sialosyl-Le^x and sialosyl-Tn structures. Each of the three lines expressed low quantities of Le^x and Tn, and did not express T. In contrast, expression of sialosyl-dimeric Le^x was high on HAL-8, moderate on HAL-33, and low on HAL-24.

C. Inhibition of Metastasis by Sialidase Treatment of Cells

Cells were detached using 2 mM EDTA in PBS, washed, and resuspended in 9 volumes of PBS. One ml of cell suspension was incubated 5 minutes at 37°C with 0.2 U/ml of Clostridium perftingens sialidase (type X, Sigma Chemical Co., St Louis, MO). After incubation, cells were washed three times, resuspended with RPMI 1640, and investigated for MP and expression of sialosyl- dimeric Le^x. MP of HAL-8 and -33 was completely inhibited by sialidase treatment of cells (see Table 2 below). Expression of sialosyl-dimeric Le^x appears to play an important role in blood-borne metastasis.

a Nude mice were injected (2 x 10⁵ cells) in the tail vein. 56 days after injection, mice were killed and metastatic nodules on lung surface were counted under dissecting microscope.

b Mean of 6 animals (range in parentheses).

From the foregoing, it will be evident that although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Claims

1. An agent selected from the group consisting of tumor-associated carbohydrate antigens that exhibit differential prognostic significance, antibodies that specifically bind to said antigens, oligosaccharide components of said antigens, and conjugates of said antigens or said oligosaccharides for use within the manufacture of a medicament for inhibiting tumor cell metastasis potential in a warm-blooded animal.

2. The agent of claim 1 wherein the oligosaccharide component is selected from the group consisting of lactose, lacto-N-tetrose, methyl D-lactoside, and phenyl D-thiolactoside.

3. The agent of claim 1 wherein the tumor-associated carbohydrate antigen is selected from the group consisting of sialosyl-Le^x, H/Le^y/Le^b, sialosyl-Le^a, sialosyl-Tn, dimeric Le^x, sialosyl-dimeric Le^x, and trifucosyl Le^x.

4. The agent of claim 1 wherein the oligosaccharide component is the oligosaccharide group of a tumor-associated carbohydrate antigen selected from the group consisting of H/Le^y/Le^b, sialosyl-Le^x, sialosyl-Le^a, sialosyl-Tn, dimeric Le^x, sialosyl-dimeric Le^x, and trifucosyl Le^x.

5. The agent of either of claims 2 or 4 wherein the oligosaccharide is coupled to poly(ethylene glycol).

6. A conjugate comprising an oligosaccharide coupled to poly(ethylene glycol).

7. The conjugate of claim 6 wherein the oligosaccharide is selected from the group consisting of lactose, lacto-N-tetrose, methyl D -lactoside, and phenyl D- thiolactoside.

8. The conjugate of claim 6 wherein the oligosaccharide is the oligosaccharide portion of a tumor-associated carbohydrate antigen selected from the group consisting of sialosyl-Le^x, H/Le^y/Le^b, sialosyl-Le^a, sialosyl-Tn, dimeric Le^x, sialosyl-dimeric Le^x, and trifucosyl Le^x.

9. A method for inhibiting tumor cell metastasis potential within a biological preparation, comprising:

incubating the biological preparation with at least one agent selected from the group consisting of tumor-associated carbohydrate antigens that exhibit differential prognostic significance, antibodies that specifically bind to said antigens, oligosaccharide components of said antigens, and conjugates of said antigens or said oligosaccharides, said agent inhibiting the metastasis potential of the preparation.

10. The method of claim 9 wherein the oligosaccharide component is selected from the group consisting of lactose, lacto-N-tetrose, methyl D-lactoside, and phenyl D-thiolactoside.

11. The method of claim 9 wherein the tumor-associated carbohydrate antigen is selected from the group consisting of sialosyl-Le^x, H/Ley/Le^b, sialosyl-Le^a, sialosyl-Tn, dimeric Le^x, sialosyl-dimeric Le^x, and trifucosyl Le^x.

12. The method of claim 9 wherein the oligosaccharide component is the oligosaccharide group of a tumor-associated carbohydrate antigen selected from the group consisting of H/Le^y/Le^b, sialosyl-Le^x , sialosyl-Le^a, sialosyl-Tn, dimeric Le^x, sialosyl-dimeric Le^x, and trifucosyl Le^x.

13. The method of claims 10 or 12 wherein the oligosaccharide is coupled to poly(ethylene glycol).