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WO1997031653A9 - Diagnostic et traitement du carcinome metastatique du sein - Google Patents

Diagnostic et traitement du carcinome metastatique du sein

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Publication number
WO1997031653A9
WO1997031653A9 PCT/US1996/013691 US9613691W WO9731653A9 WO 1997031653 A9 WO1997031653 A9 WO 1997031653A9 US 9613691 W US9613691 W US 9613691W WO 9731653 A9 WO9731653 A9 WO 9731653A9
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WIPO (PCT)
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cells
integrin
polypeptide
levels
laminin
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PCT/US1996/013691
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English (en)
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WO1997031653A1 (fr
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  • DIAGNOSIS AND TREATMENT OF METASTIC BREAST CARCINOMA The field of the invention is cancer diagnosis and therapy.
  • the invention provides methods of predicting the likelihood of metastic breast carcinom and methods for predicting the prognosis for patients diagnosed with breast carcinoma.
  • the invention also provides methods for preventing and treating breast carcinoma by blocking ⁇ 601 function.
  • the invention features a method of diagnosing a mammal for the presence of a metastatic breast cancer or an increased likelihood of developing metastatic breast cancer.
  • the method involves measuring ⁇ 6/31 integrin in a breast tissue sample from the mammal, with an increase in ⁇ 6/31 levels relative to a sample from an unaffected individual being an indication that the mammal has a malignancy or has an increased likelihood of developing a malignancy.
  • the increase in ⁇ 6j31 integrin levels is two-fold or greater.
  • kits for carrying out the above methods are also included in the invention.
  • Such kits preferably include a substantially pure antibody that specifically recognizes and binds a ⁇ 6/31 polypeptide, and may also include means for detecting and quantitating antibody binding.
  • the kit may include all or a fragment of a x6 ⁇ l nucleic acid sequence useful for hybridization purposes, and may also include means for detecting and quantitating ⁇ 601 RNA hybridization.
  • the invention also features a method of treating a mammal with a ⁇ 6 / 81-associated malignancy involving administering to the mammal a ⁇ 63l antibody in an amount sufficient to inhibit growth of the malignancy, and further features a therapeutic composition having as an active ingredient a ⁇ l antibody, formulated in a physiologically-acceptable carrier.
  • the antibody is directed to the extracellular domain of ⁇ 6)81 integrin.
  • the invention features methods of identifying ⁇ 631 inhibitory compounds.
  • the first method involves the identification of inhibitory compounds that are capable of decreasing the expression of a ⁇ 6/31 gene, involving (a) providing a cell expressing the ⁇ 6 ⁇ l gene; and (b) contacting the cell with a candidate compound, an increase in ⁇ 6/31 expression following contact with the candidate compound identifying a modulatory compound.
  • the second method involves the identification of modulatory compounds which are capable of decreasing ⁇ 6/3l function, involving (a) providing a cell expressing the ⁇ 6/31 protease; and (b) contacting the cell with a candidate compound, an decrease in ot ⁇ l activity following contact with the candidate compound identifying a modulatory compound.
  • the invention features a method of treating a mammal with a disease involving increased presence of a ⁇ 6j81, involving administering to the patient a modulatory compound (for example, identified according to the above methods) in an amount effective to reduce the symptoms of the disease in the mammal.
  • a modulatory compound for example, identified according to the above methods
  • ⁇ 6/31 polypeptide is meant an amino acid sequence which is an integrin laminim receptor as described, for example, in Mercurio, Trends Cell Biol. 5:419-423, 1993, whose expression correlates with the presence of malignant breast carcinoma.
  • a polypeptide has an amino acid sequence which is at least 45%, preferably 60%, and most preferably 85% or even 95% identical to the amino acid sequence referenced in Mercurio (1993) , supra.
  • substantially identical polypeptide sequence an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, e.g. , as described herein) .
  • conservative amino acid substitutions for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, e.g. , as described herein) .
  • such a sequence is at least 85%, more preferably 90%, and most preferably 95% identical at the amino acid level to the sequence of Fig. 10 (SEQ ID NO: 1) .
  • the length of comparison sequences will generally be at least 15 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids.
  • Homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) .
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • protein or “polypeptide” is meant any chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation or phosphorylation) .
  • substantially pure is meant a preparation which is at least 60% by weight (dry weight) the compound of interest, e.g., the ⁇ 6/3l polypeptide or ⁇ 631-specific antibody.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • purified DNA DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g. , a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences.
  • nucleic acid sequence which encodes a polypeptide differing only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, e.g., as described herein) .
  • conservative amino acid substitutions for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, e.g., as described herein) .
  • the encoded sequence is at least 45%, more preferably 60%, and most preferably 85% identical at the amino acid level to the sequence of Fig. 10 (SEQ ID NO: 1) .
  • a "substantially identical" nucleic acid sequence is one which is at least 85%, more preferably 90%, and most preferably 95% identical to the sequence of Fig. 11 (SEQ ID NO: 2) .
  • the length of nucleic acid sequence comparison will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
  • purified antibody is meant antibody which is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, antibody.
  • ⁇ 631 polypeptide By “specifically binds” is meant an antibody which recognizes and binds a ⁇ 631 polypeptide but which does not substantially recognize and bind other molecules in a sample (e.g., a biological sample) which naturally includes ⁇ 631 polypeptide.
  • a sample e.g., a biological sample
  • ⁇ 601 specifically binds
  • ⁇ 601 is sufficient to detect a ⁇ 631 protein product in such a biological sample using one or more of the standard immunological techniques available to those in the art (for example, Western blotting or immunoprecipitation) .
  • malignancy is meant any abnormal tissue that grows by cellular proliferation more rapidly than normal or that continues to grow after growth stimuli cease.
  • a malignancy according to the invention is generally either locally invasive or metastatic.
  • relative to a wild-type sample is meant either (a) relative to an equivalent tissue sample from an unaffected individual or (b) relative to an unaffected sample of similar tissue type from the mammal being diagnosed.
  • immunological methods any assay involving antibody-based detection techniques including, without limitation, Western blotting, immunoprecipitation, and direct and competitive ELISA and
  • ⁇ 6j31 RNA messenger RNA transcribed from a ⁇ 6/31 DNA sequence.
  • hybridization techniques any detection assay involving specific interactions (based on complementarity) between nucleic acid strands, including DNA-DNA, RNA-RNA, and DNA-RNA interactions. Such hybridization techniques may, if desired, include a PCR amplification step.
  • a “modulatory compound” is meant any compound capable of either decreasing ⁇ 6/Sl expression (i.e., at the level of transcription, translation, or post-translation) or decreasing c.6/91 protein activity (i.e., the amount of activity per unit of c.6/91 protein) .
  • a “differentiation agent” is meant any compound which, when added to cells in. vitro or introduced into a mammal, result in a change in the phenotype of a cell or tissue, including the expression of one or more markers indicative of a particular stage in the cell's or tissue's life cycle.
  • Differentiation agents include, without limitation, retinoic acid and cyclic AMP.
  • Fig. 1 is a graph of surface expression of integrin subunits on MDA MB-435 cells.
  • MDA MB-435 cells were analyzed by flow cytometry using MAbs specific for the indicated integrin subunits. The left scan in each profile was obtained using a nonspecific mouse IgG, and the right scan corresponds to the expression levels of the specified integrin subunits.
  • Figs. 2A and 2B are graphs of mAb inhibition of MDA MD-435 adhesion and migration on laminin and collagen I.
  • 2A shows adhesion to tissue culture wells were coated with laminin (20 ⁇ g/ml) or collagen 1 (20 ⁇ g/ml) .
  • MDS MB-435 cells were preincubated with antibodies to specific integrin subunits before addition to the protein-coated wells. After a 30 in. incubation at 37°C, nonadherent cells were removed by washing, and adherent cells were fixed, stained, and quantitated as described below. The data shown are the mean values +SEM (bars) of three experiments done in duplicate.
  • 2B shows migration data for MDA MB-435 cells preincubated with antibodies to specific integrin subunits before addition to Transwell chambers.
  • Media containing either laminin (15 ⁇ g/ml) or collagen I ( ⁇ g/ml) were added to the bottom wells. After a 4 hr. incubation at 37°C, the cells that had not migrated were removed, and the cells that had migrated onto the lower surface of the filter were fixed, stained, and quantitated.
  • the data shown are the mean values +SEM (bars) from two experiments done in duplicate, laminin; collagen I.
  • Figs. 3A-3C depict expression of the truncated B4- ⁇ CYT integrin subunits in MDA MB-435 cells.
  • Fig. 3A is a schematic demonstration of the strategy used to knock out ⁇ 631. This strategy is based on the finding that ⁇ 6 associates preferentially with /84 in comparison with ⁇ l . Thus, expression of the /34- ⁇ CYT subunit will result in formation of the ⁇ 6/34- ⁇ CYT heterodimer at the expense of C.6J1 association. Because of its cytoplasmic domain deletion, the ⁇ 6/94- ⁇ CYT integrin should be a null receptor unable to mediate laminin interactions.
  • Fig. 3B show profiles obtained when a population of transfected MDA MB-435 cells expressing 34- ⁇ CYT on the cell surface were isolated by FACS using UM-A9, a mAb specific for the ⁇ l integrin subunit. The resulting cells were analysed by flow cytometry. The left scan in each profile was obtained using a nonspecific mouse IgG, and the right scan corresponds to the expression levels of the specified integrin subunits, Mock, MDA MB-435 cells transfected with the vector alone: 34- ⁇ CYT, MDA MB-435 cells transfected with the 04- ⁇ CYT cDNA.
  • Fig. 3B show profiles obtained when a population of transfected MDA MB-435 cells expressing 34- ⁇ CYT on the cell surface were isolated by FACS using UM-A9, a mAb specific for the ⁇ l integrin subunit. The resulting cells were analysed by flow cytometry. The left scan in each profile was obtained using a nonspecific mouse IgG
  • 3C shows immunoprecipitation of transfected cells.
  • the cells were surface labeled with biotin, and aliquots of detergent extracts from equal numbers of cells were immunoprecipitated with either the 2B7 or UM-A9 mAbs.
  • Immunoprecipicies were resolved by 8% SDS-PAGE under reducing conditions and transferred to nitrocellulose filters. Proteins were visualized with streptavidin conjugated to horseradish peroxidase and enhanced chemiluminescence.
  • FIG. 4A is a graph of adhesion of MDA MB-435 transfectants. Cells were assayed for their ability to adhere to laminine and collagen I subunits. Transfected cells (109) were added to la inin- coated (20 ⁇ g/ml) and collagen I-coated (20 ⁇ g/ml) wells and incubated for 30 min. at 37°C. Nonadherent cells were removed by washing, and subsequent cells were fixed, stained and quantitated. The data shown are the mean values +SEM (bars) of these experiments done in triplicate.
  • Fig. 4B is a graph of migration of MDA MB- 435 transfectants. Cells were assayed for their ability to migrate toward laminin and collagen I.
  • Fig. 4C is a graph of invasion by MDA MB-435 transfectants. Cells were assayed for their ability to invade Marrigel.
  • Marrigel was diluted in cold distilled water, added to the upper well of Transwell chambers and dried under a sterile hood. The Marrigel was reconstructed with medium, and the transfectants (2 x 109) were added to each well. Conditioned NIH-3T3 medium was added to the bottom wells of the chambers. After 5-7 hr. at 37°C, the cells that had not invaded were removed, and the cells that had invaded to the lower surface of the filters were fixed, stained and quantitated as described in "Materials and Methods" .
  • the conclusion that the ⁇ 6 integrin is diminished in breast cancers may be misleading, because it is based on comparing the staining intensity of diverse populations of cells.
  • a second factor is that the pathological specimens used in such studies are often chosen at random, with no reference to either disease stage or other clinical parameters.
  • ⁇ 6j34 integrin can be used as a diagnostic and prognostic antibody.
  • therapies which eliminate ⁇ 631 from the cell surface or block ⁇ 631 function may be used to slow, halt, or prevent metastisis of breast cancer.
  • the following examples are provided to illustrate the invention. They are not intended to limit the scope of the invention.
  • Anti-c.6fil Antibodies are provided to illustrate the invention. They are not intended to limit the scope of the invention.
  • a ⁇ 6j81 coding sequence i.e., the whole protein or the ctyoplasmic region may be expressed as a C-terminal fusion with glutathione S-transferase (GST) (Smith et al., Gene 67:31-40, 1988).
  • GST glutathione S-transferase
  • the fusion protein may be purified on glutathione-Sepharose beads, eluted with glutathione cleaved with thrombin (at the engineered cleavage site) , and purified to the degree necessary for immunization of rabbits.
  • Primary immunizations may be carried out with Freund's complete adjuvant and subsequent immunizations with Freund's incomplete adjuvant.
  • Antibody titres are monitored by Western blot and immunoprecipitation analyses using the thrombin- cleaved ⁇ 6)81 protein fragment of the GST- ⁇ 631 fusion protein.
  • Immune sera may be affinity purified using CNBr-Sepharose-coupled ⁇ 631 protein.
  • Antiserum specificity is determined using a panel of unrelated GST proteins (e.g., GSTp53, Rb, HPV-16 E6, and E6-AP) and GST-trypsin (which may be generated by PCR using known sequences) .
  • peptides corresponding to relatively unique hydrophilic regions of a ⁇ l may be generated and coupled to keyhole limpet hemocyanin (KLH) through an introduced C-terminal lysine.
  • KLH keyhole limpet hemocyanin
  • Antiserum to each of these peptides is similarly affinity purified on peptides conjugated to BSA, and specificity tested in ELISA and Western blots using peptide conjugates, and by Western blot and immunoprecipitation using ⁇ 601 expressed as a GST fusion protein.
  • monoclonal antibodies may be prepared using the ⁇ 631 proteins described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981; Ausubel et al., supra) .
  • monoclonal antibodies are also tested for specific ⁇ 6/31 recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra) .
  • Antibodies which specifically recognize ⁇ 6/31 are considered to be useful in the invention; such antibodies may be used, e.g., in an immunoassay to monitor the level of ⁇ 6/9l produced by a mammal (for example, to determine the amount or location of ⁇ 6j81) .
  • antibodies of the invention are produced using fragments of the ⁇ 6)Bl protein which lie outside highly conserved regions and appear likely to be antigenic, by criteria such as high frequency of charged residues.
  • fragments are generated by standard techniques of PCR and cloned into the pGEX expression vector (Ausubel et al., supra) . Fusion proteins are expressed in E_j_ coli and purified using a glutathione agarose affinity matrix as described in Ausubel et al. (supra) .
  • two or three such fusions are generated for each protein, and each fusion is injected into at least two rabbits. Antisera are raised by injections in a series, preferably including at least three booster injections.
  • c.6/31 cDNA facilitates the identification of molecules which increase or decrease ⁇ 6 ⁇ l expression.
  • candidate molecules are added at varying concentrations to the culture medium of cells expressing c.6/31.
  • c.6/31 expression is then measured, for example, by standard Northern blot analysis (Ausubel et al. , supra) using a c.6/31 cDNA (or cDNA fragment) as a hybridization probe.
  • the level of c.6/31 expression in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium but in the absence of the candidate molecule.
  • the effect of candidate modulators on expression may, in the alternative, be measured at the level of c.6/31 protein production using the same general approach and standard immunological detection techniques, such as Western blotting or immunoprecipitation with a ⁇ 63l-specific antibody (for example, the c.6/31 antibody described herein) .
  • Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells; Ausubel et al., supra) .
  • a mixed compound assay c.6/81 expression is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques, e.g., HPLC or FPLC) until a single compound or minimal compound mixture is demonstrated to modulate c.6/31 expression.
  • Candidate c.6/31 modulators include peptide as well as non-peptide molecules (e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured) .
  • non-peptide molecules e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured
  • a molecule which promotes a decrease in c.6/31 expression or c.6/31 activity is considered particularly useful in the invention; such a molecule may be used, for example, as a therapeutic to dencrease cellular levels of c.6/31.
  • Modulators found to be effective at the level of c.6/31 expression or activity may be confirmed as useful in animal models and, if successful, may be used as anti- cancer therapeutics.
  • a c.6/31 modulator may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer a ⁇ l to patients suffering from or presymptomatic for a ⁇ 6/31- associated carcinoma.
  • Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for c.6/31 modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • treatment with a c.6/31 modulatory compound may be combined with more traditional cancer therapies such as surgery, radiation, or chemotherapy.
  • Detection of A Malignant Condition ⁇ t6j81 polypeptides and nucleic acid sequences find diagnostic use in the detection or monitoring of breast carcinoma.
  • a increase in the level of ⁇ 601 production provides an indication of a malignant or pre-malignant condition.
  • Levels of c.6/31 expression may be assayed by any standard technique. For example, its expression in a biological sample (e.g.
  • a biopsy may be monitored by standard Western blot analysis or may be aided by PCR (see, e.g., Ausubel et al., supra; PCR Technology: Principles and Applications for DNA Amplification, ed. , H.A. Ehrlich, Stockton Press, NY; and Yap and McGee, Nucl. Acids. Res. 19:4294, 1991).
  • immunoassays are used to detect or monitor c.601 protein in a biological sample.
  • ⁇ 6 / 31-specific polyclonal or monoclonal antibodies produced as described above may be used in any standard immunoassay format (e.g. , ELISA, Western blot, or RIA assay) to measure c.6/31 polypeptide levels; again comparison is to wild-type c.6/31 levels, and a decrease in c.6/31 production is indicative of a malignant condition.
  • standard immunoassay format e.g., ELISA, Western blot, or RIA assay
  • Examples of immunoassays are described, e.g., in Ausubel et al., supra. Immunohistochemical techniques may also be utilized for c.6/81 detection.
  • a tissue sample may be obtained from a patient, and a section stained for the presence of c.6/31 using an anti-a ⁇ l antibody and any standard detection system (e.g., one which includes a secondary antibody conjugated to horseradish peroxidase) .
  • any standard detection system e.g., one which includes a secondary antibody conjugated to horseradish peroxidase
  • Bancroft and Stevens Theory and Practice of Histological Techniques, Churchill Livingstone, 1982
  • Ausubel et al. (supra) .
  • the methods of the instant invention may be used to reduce or diagnose the disorders described herein in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated or diagnosed, the c.6/31 polypeptide, nucleic acid, or antibody employed is preferably specific for that species.
  • the MDA-MB-435 breast carcinoma cell line was obtained from the Lombardi Breast Cancer Depository (Georgetown University) .
  • the cells were grown in DMEM (GIBCO) supplemented with 10% FCS (GIBCO) and 1% penicillin-streptomycin (GIBCO) .
  • the antibodies specific for integrin subunits were as follows: mAb 3 13 (/31) ; UM-A9 (04) ; IIE10 ( ⁇ 2) ; IVA5 ( ⁇ 3) ; and 2B7 ( ⁇ 6) (Shaw et al., J. Biol . Che . 268:11401-11408, 1993); and Mouse IgG was obtained from Sigma Chemical Co.
  • the stable transfectants were pooled, and a population of cells that expressed the human ⁇ A subunit on the cell surface was isolated by FACS.
  • the sorting was repeated sequentially to enrich for homogeneous populations of cells expressing high levels of the transfected /34- ⁇ CYT subunit on the cell surface.
  • Transfected MDA-MB-435 cells were washed twice with RPMI 1640 media containing 25 mm HEPES (RPMI-H) and 0.2% BSA (RH/BSA). Aliqouts of cells (5 x 10 5 ) were incubated for 45-60 min at room temperature with RH/BSA containing UM-A9 (3 ⁇ g/ml) . The cells were washed two times with RH/BSA and then incubated with goat F(ab') 2 antimouse IgG coupled to FITC (Iago) for 45-60 min at room temperature. After washing two times with RH/BSA, the cells were resuspended in the same buffer and analyzed using a FACSc (Becton Dickinson) .
  • FACSc Becton Dickinson
  • Adhesion assays were performed as described previously (Shaw et al., J. Biol . Chem . 268:11401-11408, 1993). Briefly, ultiwell tissue culture plates (11.3-mm diameter) were coated overnight at 4°C with 0.2 ml PBS (20 ⁇ g/ml) containing either murine Engelbreth-Holm-Swain laminin or rat collagen I. The wells were then washed with PBS, and 10 5 cells in RPMI-H containing 1% DSA were added.
  • Cell migration assays were performed using 6.5-mm Transwell chambers (8- ⁇ m pore size; Costar) as described previously (Shaw and Mercurio, Mol . Biol . Cell 5:679-690, 1994). Briefly, RPMI-H containing 15 ⁇ g/ml laminin (0.6 ml) was added to the bottom well, and the filters were coated for -30 min at 37°C. Cells were resuspended in the appropriate buffer at a concentration of 10 5 /ml, and 10 5 cells were added to the top well of the Transwell chambers.
  • the cells were resuspended in the same buffer at a concentration of 5 x 10 5 cells/ml, NHS- LC-biotin (Pierce Chemical Co., Rockville, IL) was resuspended in DMSO and added to the cells at a concentration of 0.1 mg/ml. Cells were incubated in the presence of biotin for 1 hour at room temperature with gentle shaking. Subsequently, the cells were washed several times with PBS containing 50 mM NH 4 C1 to remove unincorporated biotin.
  • Immune complexes were recovered with protein G agarose (Pharmacia) .
  • the agarose beads were added for 1 h at 4°C with constant agitation.
  • the cell extracts were immunoprecipitated initially with /34-specific antibodies, and the supernatant was immunoprecipitated subsequently with ⁇ 6- specific antibodies.
  • the beads were washed two times with 50 mM Tris buffer (pH 7.5) containing 0.15M NaCl and 0.1% Triton X-100. two times with the same buffer containing 0.5 M NaCl, and one time with 0.05 M Tris (pH 6.8) .
  • Laem eli sample buffer was added to the samples, which were then incubated at 100°C for 4 min.
  • MDA-MB-435 cells were analyzed by flow cytometry to determine the cell surface expression of integrin laminin receptor subunits. As shown in Fig. 1, these cells express the ⁇ 2, ⁇ 3 , ⁇ 6, and ⁇ l subunits, but they lack expression of the ⁇ A subunit. These data indicate that three possible integrin laminin receptors are expressed on the cell surface by this cell line: c.2jBl, c.3/31, and c.6/31. However, expression of an integrin receptor on the cell surface does not always correlate with functional activity (Mercurio, Trends. Cell Biol. 5:419-423, 1995).
  • adhesion assays were performed in the presence of inhibitory antibodies specific for each of the integrin subunits. Antibodies that recognize the ⁇ 6 and ⁇ l subunits inhibited adhesion to laminin almost completely (Fig. 2A) . In contrast, function-blocking antibodies specific for the ⁇ 2 and ⁇ 3 subunits had no inhibitory effect on laminin adhesion compared with the control mouse IgG antibody. The ⁇ 2-specific MAb did inhibit adhesion to collagen I, however (Fig. 2A) .
  • MDA-MB-435 cells were transfected with the /34- ⁇ CYT cDNA and a population of stably transfected cells that expressed high surface levels of ⁇ A were obtained by FACS (Fig. 3B) .
  • the total level of ⁇ 6 on the cell surface was not altered in the /34- ⁇ CYT transfectants (Fig. 3B) .
  • the amount of ⁇ 6/31 receptor present on the cell surface was decreased by the expression of the /84- ⁇ CYT CDNA, as shown in Fig.
  • MDA-MB-435 cells that had been transfected with either the vector alone or the /84-f/CYT cDNA were analyzed for their ability to adhere and migrate on laminin. As shown in Figs. 4A and 4B, cells that expressed the truncated ⁇ A subunit were inhibited almost completely in their ability to both adhere and migrate on laminin. In contrast, their adhesion and migration on collagen I did not differ significantly from the mock transfectants, indicating that the inhibition wa specific for laminin. In control experiments, the mock and /34- ⁇ CYT transfectants also adhered comparably to uncoated Transwell filters.
  • the dominant-negative transfection strategy described in this study should be a powerful tool for knocking out the function of c.6/31 specifically in other cell types that do not express a ⁇ A .
  • Such an approach could be extremely useful, because c.6/31 has been implicated in a myriad of biological and pathological phenomena (Mercurio et al., Trends, Cell Biol . , 5:419-423, 1995).
  • expression of this dominant-negative receptor can be used to dissect the functions of ⁇ 6/3l in cells that express multiple integrin laminin receptors.
  • RKO rectal carcinoma cells use both the c.6/31 and ⁇ 2/Sl receptors for mediating laminin interactions.
  • the invention also includes use of antibodies to ⁇ 6/81 polypeptide fragments.
  • fragment means at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids.
  • Fragments of c.6/31 polypeptides can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events) .
  • Preferable fragments or analogs according to the invention are those which facilitate specific detection of a c.6/31 polypeptide or polypeptide levels in a sample to be diagnosed.

Abstract

La présente invention présente une méthode de détection de l'accroissement de la probabilité du cancer métastatique du sein par la détection des niveaux de l'intégrine α6β1. Elle présente également des méthodes de prévention ou de traitement du cancer métastatique du sein par blocage de la fonction α6β1.
PCT/US1996/013691 1996-03-01 1996-08-23 Diagnostic et traitement du carcinome metastatique du sein WO1997031653A1 (fr)

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US1264396P 1996-03-01 1996-03-01
US60/012,643 1996-03-01

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JP4912528B2 (ja) 1998-04-02 2012-04-11 キシンテラ、アクチボラグ インテグリンヘテロ二量体およびそのサブユニット
SE0301087D0 (sv) 2003-04-14 2003-04-14 Cartela Ab New monoclonal antibody

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