WO1992004048A2 - Inhibition du potentiel metastatique et du caractere envahissant de cellules tumorales grace a l'utilisation d'oligosaccharides et d'antigenes ou d'anticorps d'oligosaccharides - Google Patents

Inhibition du potentiel metastatique et du caractere envahissant de cellules tumorales grace a l'utilisation d'oligosaccharides et d'antigenes ou d'anticorps d'oligosaccharides Download PDF

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WO1992004048A2
WO1992004048A2 PCT/US1991/006202 US9106202W WO9204048A2 WO 1992004048 A2 WO1992004048 A2 WO 1992004048A2 US 9106202 W US9106202 W US 9106202W WO 9204048 A2 WO9204048 A2 WO 9204048A2
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sialosyl
oligosaccharide
tumor
antigens
group
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PCT/US1991/006202
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WO1992004048A3 (fr
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Tatsushi Toyokuni
Sen-Itiroh Hakomori
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The Biomembrane Institute
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Priority to AU86677/91A priority Critical patent/AU659808B2/en
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Publication of WO1992004048A3 publication Critical patent/WO1992004048A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine

Definitions

  • 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.
  • cancer 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.
  • host tissue barriers such as the epithelial basement membrane
  • the present invention provides a variety of agents and methods for inhibiting metastasis potential and invasiveness of tumor cells.
  • a method for inhibiting tumor cell metastasis potential within a biological preparation 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.
  • 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).
  • oligosaccharides for use within the present invention include lactose, lacto-N-tetrose, methyl ⁇ -D -lactoside and phenyl ⁇ -D- thiolactoside. Oligosaccharides may be used individually or in combination with one another.
  • 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 ⁇ -D-lactoside or phenyl ⁇ -D-thiolactoside on the number and size of lung colony deposits of BL6 cells.
  • BL6 were preincubated with control medium, 0.1 M methyl ⁇ - D-lactoside ("Me- ⁇ -lactoside"), or 0.1 M phenyl ⁇ -D-thiolactoside ("phe- ⁇ -S-lactoside).
  • 2 x 10 4 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 ⁇ -D-lactoside on the number and size of lung colony deposits of BL6 cells.
  • Methyl ⁇ -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 ⁇ -D-lactoside) and groups B and C represent animals injected with 0.25 M and 0.5 M methyl ⁇ -D-lactoside, respectively.
  • column 1 represents the total number of colonies
  • column 2 the number of colonies with diameter > 1 mm
  • 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)- ⁇ -lactoside.
  • Tumor cells were intravenously injected, followed by intraperitoneal injection of: PBS control (A); 0.25 M Me- ⁇ -lactoside (B); 0.5 M Me- ⁇ -lactoside (C); 0.5 M lactose (D); 0.25 M N-acetyllactosamine (E); or 0.5 M Me- ⁇ -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 H 1 -liposome binding to various glycolipids.
  • Panel B shows Le y -liposome binding to various glycolipids.
  • 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.
  • 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.
  • TACAs tumor-associated carbohydrate antigens
  • GSL glycosphingolipid
  • 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.
  • 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).
  • TACAs may be distinguished through a comparison of invasiveness, metastasis and clinical prognosis of similar tumors showing expression vs. non-expression of such TACAs.
  • 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 .
  • 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
  • GM3 structure 9
  • TACAs for use within the present invention exhibit a differential prognostic significance.
  • 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.
  • FIG 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.
  • antibodies to suitable TACAs may also be employed within the context of the present invention.
  • 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.
  • 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.
  • MAbs monoclonal antibodies
  • 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.
  • 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
  • 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.
  • oligosaccharide components of suitable TACAs may also be used within the present invention.
  • 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 ⁇ -D -lactoside, lacto-N- tetrose (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4Glc), and phenyl ⁇ -D-thiolactoside.
  • lactose and lactose derivatives such as methyl ⁇ -D -lactoside, lacto-N- tetrose (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4Glc), and phenyl ⁇ -D-thiolactoside.
  • lactose derivatives such as methyl ⁇ -D -lactoside, lacto-N- tetrose (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4Glc), and phenyl ⁇ -D-thiolactoside.
  • Other lactose derivatives
  • 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.
  • 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).
  • 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.
  • 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.
  • GSL giycosphingolipid
  • 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.
  • 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.
  • linker group 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.
  • a carboxyl group may be activated. Activation of a carboxyl group includes formation of an "active ester," such as a succinimidyl ester.
  • active ester is known to refer to esters which are highly reactive in nucleophilic substitution reactions.
  • 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).
  • oligosaccharides may be coupled to (i.e., covalently bonded to) a straight-chain amphiphilic polymer, such as poly(ethylene glycol).
  • 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 NH 2 -(CH 2 CH 2 -O) n -CH 3 , 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 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.
  • 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.
  • 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.
  • such substances may be administered in combination with an immunotherapeutic or chemotherapeutic agent
  • 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.
  • Heptaacetyllactosylimidate (Zimme ⁇ nann 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 ⁇ -D-lactoside in 68% yield from the imidate: m.p. 211-212°C (lit.
  • Lactose octaacetate (Hudson and Kunz, J. Am. Chem. Soc. 47:2052, 1926) was treated with thiophenol and SnCl 4 (Nicolaou et al., J. Am. Chem. Soc. 110:7910. 1988) in dichloromethane at 0°C to give phenyl heptaacetyl ⁇ -D-thiolactoside in 80% yield. This product was deacetylated with NaOMe in MeOH and neutralized with Amberlyst ® 15.
  • This oligosaccharide (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1-4Glc) was prepared from human milk by pretreatment with ethanol and recycling BioGel P-2 column chromatography with water as eluent, followed by reversed-phase (C 18 ) high pressure liquid chromatography with water (Dua and Bush, Anal. Biochem. 133:1. 1983).
  • the 1 H-NMR spectrum was superimposed on that of the authentic sample (BioCarb Chemicals, Lund, Sweden).
  • Poly(ethylene glycol) derivative of ⁇ -D-lactoside The reaction scheme is as follows: The poly(ethylene glycol) derivative of ⁇ -D-lactoside was prepared from readily available 3-succinimidooxycarbonylpropyl O-(2, 3, 4, 6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)-(1 ⁇ 4)-2,3,6-tri-O-acetyl- ⁇ -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).
  • 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
  • a suspension of BL6 cells (1-3 x 10 6 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 10 4 or 2 x 10 4 cells (with or without oligosaccharide pretreatment) per 200 ⁇ l 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.
  • ANOVA analysis of variance
  • BL6 cells were incubated with various concentrations of lactose, lacto-N-tetrose (Gal ⁇ 1 ⁇ 3GlcNAc ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4Glc), methyl ⁇ -D-lactoside, or phenyl ⁇ -D-thiolactoside for various durations.
  • lactose lacto-N-tetrose
  • methyl ⁇ -D-lactoside or phenyl ⁇ -D-thiolactoside
  • 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 ⁇ -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 ⁇ -D-lactoside was reproduced in three separate experiments and the reduction was found to be consistently between 35% and 45%.
  • the inhibitory effect on tumor deposition is not related to the effect on cell growth in vitro or in vivo.
  • mouse melanoma B16 variants showing different degrees of metastatic potential 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.
  • 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.
  • 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% CO 2 /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 ⁇ l 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.
  • HAL-8, HAL-24, and HAL-33 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.
  • 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.
  • mice were injected (2 x 10 5 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).
  • 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 ⁇ g/ml of immunoglobulin.
  • MAb SH1 monoclonal antibodies directed to Le x
  • MAb SH4 sialosyl-Le x
  • MAb FH6 sialosyl-dimeric Le x
  • T MAb HH8
  • Tn MAb 1E3
  • sialosyl-Tn MAb TKH2
  • 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 .
  • 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.
  • expression of sialosyl-dimeric Le x was high on HAL-8, moderate on HAL-33, and low on HAL-24.
  • mice were injected (2 x 10 5 cells) in the tail vein. 56 days after injection, mice were killed and metastatic nodules on lung surface were counted under dissecting microscope.

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Abstract

L'invention se rapporte à des agents et à des procédés destinés à inhiber le potentiel métastatique de cellules tumorales. On peut inhiber le potentiel métastatique de cellules tumorales en utilisant un agent choisi dans le groupe composé: (a) d'antigènes d'hydrates de carbone associés à la tumeur (TACA) qui possèdent une importance prognostique différentielle; (b) d'anticorps qui se lient spécifiquement à ces antigènes; (c) de constituants oligosaccharides de ces antigènes; et (d) de conjugués de ces antigènes ou de ces oligosaccharides, l'agent ainsi formé ayant le pouvoir d'inhiber le potentiel métastatique de la préparation. Des oligosaccharides supplémentaires utilisables dans ces procédés et dans ces compositions sont constitués par le lactose et par des dérivés de lactose. L'invention décrit également des conjugués comprenant des oligosaccharides et du poly(éthylène glycol).
PCT/US1991/006202 1990-08-30 1991-08-29 Inhibition du potentiel metastatique et du caractere envahissant de cellules tumorales grace a l'utilisation d'oligosaccharides et d'antigenes ou d'anticorps d'oligosaccharides WO1992004048A2 (fr)

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Cited By (7)

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WO1993000588A1 (fr) * 1991-06-26 1993-01-07 The Biomembrane Institute Diagnostic precoce du cancer du poumon grace a des combinaisons d'anticorps anti-hydrates de carbone
EP0648239A1 (fr) * 1992-07-02 1995-04-19 Collagen Corporation Conjugues polymeres biocompatibles
AU669218B2 (en) * 1992-10-27 1996-05-30 Behringwerke Aktiengesellschaft Prodrugs, their preparation and use as pharmaceuticals
WO1996019243A1 (fr) * 1994-12-21 1996-06-27 Co Enzyme Technology Ltd. Composition pour le traitement de tumeurs malignes et de leurs metastases
US5550188A (en) * 1988-11-21 1996-08-27 Collagen Corporation Polymer conjugates ophthalmic devices comprising collagen-polymer conjugates
EP0852243A1 (fr) * 1995-04-14 1998-07-08 Kazunori Kataoka Oxydes de polyethylene ayant un groupe saccharide a une extremite et un groupe fonctionnel different a l'autre extremite, et procede pour produire lesdits oxydes de polyethylene
US6540999B1 (en) 1996-01-31 2003-04-01 President And Fellows Of Harvard College Immunomodulatory methods using Lewisx oligosacchardies

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5550188A (en) * 1988-11-21 1996-08-27 Collagen Corporation Polymer conjugates ophthalmic devices comprising collagen-polymer conjugates
WO1993000588A1 (fr) * 1991-06-26 1993-01-07 The Biomembrane Institute Diagnostic precoce du cancer du poumon grace a des combinaisons d'anticorps anti-hydrates de carbone
US5639737A (en) * 1991-11-04 1997-06-17 Co Enzyme Technology Ltd. Method and compositions for treating malignant tumors and inhibiting growth and metastases of malignant tumors
EP0648239A1 (fr) * 1992-07-02 1995-04-19 Collagen Corporation Conjugues polymeres biocompatibles
EP0648239A4 (fr) * 1992-07-02 1995-09-27 Collagen Corp Conjugues polymeres biocompatibles.
AU669218B2 (en) * 1992-10-27 1996-05-30 Behringwerke Aktiengesellschaft Prodrugs, their preparation and use as pharmaceuticals
WO1996019243A1 (fr) * 1994-12-21 1996-06-27 Co Enzyme Technology Ltd. Composition pour le traitement de tumeurs malignes et de leurs metastases
AU692021B2 (en) * 1994-12-21 1998-05-28 Co Enzyme Technology Ltd. Composition for treatment of malignant tumors and their metastases
EP0852243A1 (fr) * 1995-04-14 1998-07-08 Kazunori Kataoka Oxydes de polyethylene ayant un groupe saccharide a une extremite et un groupe fonctionnel different a l'autre extremite, et procede pour produire lesdits oxydes de polyethylene
EP0852243A4 (fr) * 1995-04-14 1998-08-26 Kataoka Kazunori Oxydes de polyethylene ayant un groupe saccharide a une extremite et un groupe fonctionnel different a l'autre extremite, et procede pour produire lesdits oxydes de polyethylene
US6540999B1 (en) 1996-01-31 2003-04-01 President And Fellows Of Harvard College Immunomodulatory methods using Lewisx oligosacchardies
US6841543B1 (en) 1996-01-31 2005-01-11 President And Fellows Of Harvard College Methods of inhibiting production of T helper type 2 cytokines in human immune cells
US7799755B2 (en) 1996-01-31 2010-09-21 The President And Fellows Of Harvard College Immunomodulatory methods using oligosaccharides

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