Classifications

• • 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
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

• MAMMAMODULIN A HORMONE-INDEPENDENT MAMMARY TUMOR CELLS SPECIFIC PROTEIN.
• This invention relates to mam ⁇ iamodulin, which is a novel factor not known to exist so far in nature, produced by hormone-independent human mammary tumor cells affecting the morphology, growth and hormone receptor expression of hormone-dependent tumor cells. More specifically the invention relates to mammamodulin in purified form, i.e. at least partially free from compounds with which it is associated in nature, up to high purity, e.g. enabling the determination of at least partial amino acid sequence(s). Means for the production and purification of mammamodulin are described, as are related diagnostic and therapeutic strategies made possible by the discovery of this factor.
• MM mammamodulin
• MM analogues may be used therapeutically to block MM receptors on hormone-dependent cells.
• MM is a protein having an apparent molecular weight of approximately 52-55 k ⁇ odaltons, as measured by SDS polyacrylamide gel electrophoresis of highly purified preparations on 10-15% gradient gels. MM comprises partial amino acid sequences of
• the protein as a whole may contain allelic variations comprising additions, deletions, insertions or substitutions of residues comprising up to ten percent, but preferably no more than five percent, of the complete amino acid sequence, so long as the biological activity is retained.
• the protein may also contain various post-translational modifications such as glycosylation and formation of cysteine bridges.
• MM is further characterized in that it is heat and acid labile, and trypsin- and mercaptoethanol-sensitive, and easily forms heterogeneous aggregates with other proteins.
• MM can be extracted and purified to apparent homogeneity from cell cultures of fast-growing hormone-independent tumor cells, as described in the examples herein.
• MM may also be obtained by using amino acid sequence information to screen a cDNA library from a producer cell line to obtain the cDNA of the MM gene. The gene may then be cloned and introduced into an appropriate vector for expression in a suitable prokaryotic or eukaryotic system.
• Purified MM may be used as an antigen to produce polyclonal or monoclonal antibodies by conventional processes.
• Antibodies, in particular monoclonal antibodies are useful (i) as therapeutics to block MM and prevent it from stimulating hormone-dependent cells; and (ii) in assays (including bioassays, radioimmunoassays (RIAs), fluoroimmunoassays (FIAs),
• Purified MM may also be used in a binding assay to screen and identify compounds having affinity for MM and in competitive assays to screen and identify compounds having affinity for MM receptors on hormone-dependent cells. Such compounds are useful in inhibiting the biological activity of MM.
• compounds which inhibit the expression of MM in tumor cells in particular mammary tumor cells, e.g. hormone-independent mammary tumor cells and/or have affinity for mammamodulin receptors on tumor cells, in particular on mammary tumor cells, e.g. hormone-independent mammary tumor cells are novel and useful.
• the only compound known to be a potent inhibitor of MM is heparin.
• the human, breast cancer cell line MDA-MB-231 was grown in HL medium supplemented with 5 % fetal calf serum.
• the optimized HL medium was developed for the human leukemia cell line HL60 as described in European Patent AppHcation Publication No. 417563.
• the cells were grown in an adherent fashion with a doubling time of about 22 - 24 hours in T-flasks under 5 % C ⁇ 2-balance air and 96 % water saturation. After achieving the confluent state, the growth medium was replaced by the serum-free HL medium. Mammamodulin (MM) is released into the culture medium during growth as well as in the non-growth confluent state.
• MM Mammamodulin
• a slightly modified 23 1 airlift fermentor (Chemap AG, Switzerland) was used for the scale-up production of MM .
• the outer volume of the draft tube was filled with 8.5 kg Raschig rings (8 8 5 mm, stone ware) and 10 Siraf 25 rings (25 x 25 x 2 mm, porous glass Raschig rings, Schott, Germany) giving about 20 ⁇ fi available surface area.
• the fermentor was inoculated with tryp- sinized MDA-MB-231 cells from 2 Cell Factories units (Nunc, 6000 cm 2 per unit). Two medium exchanges were performed during the growth phase of 15 days.
• the airlift fermentor was aerated with 1 normal liter /min air and the solved oxygen content (30 %), pH (7.2) and temperature (37°C) were continuously monitored.
• MM-containing spent medium was collected by changing 17 - 18 1 serum-free fortified HL medium (glucose from 5 g to 7.5 g, glutamine from 5 mM to 6.5 mM, Primatone RL from 0.25 % to 0.3 %) daily.
• the metabolic state of the cells was monitored during the whole fermentation process by measuring glucose and glutamine consumption and lactate and ammonia formation as described (Schumpp B. and Schlaeger E.-J., J. Cell. Sci. 97: 639- 647 [1990]).
• the MM titre remained constant during the production campaign.
• the MM containing cell-free fermentor volume was concentrated 20-fold (100 1 to 5.5 1) by using an Amicon SP20 ultrafiltration unit (Amicon, Switzer ⁇ land) equipped with two S10Y10 spiral-wound cartridges (1.8 m ⁇ membrane area, MW cutoff 10 kDa).
• the MM-containing concentrates were frozen at -80°C.
• Example 2 MM was purified from 130 1 of supernatant. In difference to Example 2 more MM was found in the flow through of the Q-Sepharose and this chromatography was therefore repeated with the flow through. Also further chromatography via Heparin-Sepharose and Superdex 200 was done via 2 separate runs. The gradient of the Mono S chromatography was improved as follows:
• these fractions were concentrated using an Amicon stirring cell of 10 ml equipped with an YM10 membrane. Volume was reduced from 11 ml to 0,7 ml. 2,5 ml of buffer F were added and the volume was reduced to 0,7 ml. Dilution and concentration were once repeated and the sample of 0,7 ml was applied to Superdex 200 column (1 X 30 cm, 0,5 ml/min) preequlibrated in buffer F (for chromatogram see Fig. 6). 50 ⁇ l -aliquots of the resulting fractions were transfered into buffer G by the use of a Fast Desalting column (Pharmacia) in connection with the Pharmacia SMART system.
• a Fast Desalting column Pharmacia
• the resulting samples were investigated by SDS-PAGE (reduced, 12,5 % T, Coomassie stain, see Fig. 7).
• the major fraction (m) exhibits only one protein band in the range from 41 to 45 kD.
• the yield in this fraction is 0,12 OD280, the activity 4X10 6 U and the specific activity is 33X10 6 U/OD280.
• the total yield based on activity is 36 % and the purification factor 170.000.
• MM was purified from 146 1 of supernatant with the following steps (omitting chromatography on Q-Sepharose): 6.6 1 of concentrate were diluted and loaded on a column of Heparin Sepharose (80 ml) using 1 mM CHAPS as detergent. The active 0 fractions eluted were diluted and loaded on a column of Heparin Sepharose (50 ml) using 1 mM CHAPS as detergent and 2 mM spermidine as an additive in all buffers. The active fractions were pooled, diluted with a buffer containing 4 mM zwittergent 3-12 as detergent and loaded on a column of Heparin Sepharose (10 ml).
• the active fractions (8 ml) were pooled and, after 5 supplementation with 6 M guanidinium hydrochloride, purified in 4 runs with 2 ml each on a Superdex 200 column (120 ml).
• the active fractions were pooled, diluted and concentrated in two steps using a 10 ml and a 2 ml column of Heparin Sepharose.
• the fraction with the highest activity (2 x IO- 6 U/ml) of the final step was used for growth experiments as described in 0 Example 5C).
• This fraction was run on a SDS acrylamid gradient gel (10-15%, Phast Gel, Pharmacia). After silver staining it showed a major band of approximately 52 kDa.
• Example 2 The material from Example 2 ( fractions f to h) was purified according procedure A and fraction n from Example 3 was concentrated according pro ⁇ cedure B, because of the high salt concentration (Buffer F) present in the sample.
• Procedure A The ionic strength of the material from Example 2 was re ⁇ substituted by fourfold dilution and then concentrated on a Mono S (PC 1.6/5) (Phar ⁇ macia, Uppsala, Sweden) column on a SMART chromatography system (Pharmacia).
• the column was developed with a flow rate of 100 ⁇ l / min and a linear gradient (12.5%/min) from buffer G to H (for buffer composition see Example 2). 50 ⁇ l fractions were collected and active fractions (for activity test see Ex ⁇ ample 6) were pooled. A typical chromatogram of the concentration of MM is shown in Figure 8.
• Procedure B The material from Example 3 was concentrated on a re ⁇ versed phase column Poros R H (Perseptive Biosystems, Cambridge, MA USA) (inner diameter 0.8mm, length 10 cm) packed by LC Packings, Amster ⁇ dam, Netherlands on the SMART chromatography system from Pharmacia using the following buffers:
• the column was developed with a flow rate of 50 ⁇ l / min and a linear gradient (5%/min) from buffer J to K. 25 ⁇ l fractions were collected and ac ⁇ tive fractions were pooled.
• the samples from procedures A and B were applied to agarose gels (Pro- Sieve Gel System from FMC Bioproducts, Rockland , ME, U.S.A) and run as described by the manufacturer under non-reducing conditions using high quality grade SDS.
• the agarose gel was cut into 2 ⁇ m ⁇ slices from the anode to the cathode and the protein was eluted at am ⁇ bient temperature for at least four hours in
• the pooled material was applied to a reversed phase column Poros R H (Perseptive Biosystems, Cambridge, MA, U.S.A) (inner diameter 0.8mm, length 10 cm) packed by LC Packings, Amsterdam, Netherlands) on a
• MM MM was reduced, S-carboxy-amidomethylated and digested with endoproteinase Lys-C (Wako, Neuss, Germany) for 16 hours at 37°C. Enzyme to substrate ratio was 1:100.
• the digest was collected on a Mini S cation exchanger column (3x0.5 cm) equilibrated with 20 % acetonitrile in potassium phosphate, pH 2.5. The column was developed with a NaCl gradient and the peak-containing fractions were collected.
• Peaks were further purified on Vydac C4 (0.1x15cm) or Brownlee RP-300 (0.1x10cm) reversed phase columns, developed with a water/acetonitrile-0.1 % TFA gradient and sequenced on a 475 A or 477A sequenator (Applied Biosystems, Foster City, CA). Sequences of three peptides were obtained having the amino acid sequences of Leu-Val-Leu-Arg-X-X-Glu-Thr (SEQ ID No: 1),
• X represents a sequencing cycle in which the amino acid was not possible to identify.
• Mor p holo gy The morphological changes induced by exposing ZR-75-
• the photomicrographs show that unstimulated hormone-dependent cells grow in culture as epithelial-like patches with tight intercellular contacts (Fig. 11a).
• the peripheral cells show ruffling membranes and start to produce lamellipodia (Fig. lib) within minutes of stimulation by MM-containing conditioned medium from the producer cells or by purified protein preparations of all purification stages.
• the colonies enlarge considerably due to cell flattening and cell-to-cell contacts get weaker in the following time (Figs, lie and lid).
• Activation of the responding cells by highly purified MM lasted several hours. Then, the cells reassumed the original patch-like formation. The duration of the morphological changes was dependent on the concentration of MM and lasted longer at higher concentrations.
• Cell activation was observed in a wide variety of hormone- dependent cells as shown in Table I. All cell lines in Table I are publicly available from, e.g., American Type Culture Collection (ATCC) Rockville, Maryland, USA.
• MDA-MB-453 did not respond with morphological changes upon stimulation by purified MM or by conditioned medium from MDA-MB-231 cells nor did they secrete a factor activating membrane ruffling of ZR-75-1 or T-47D cells.
• Hormone-dependent T47-D cells were seeded into 96 well cell culture plates previously coated with collagen type IV at a density of 10,000 cells per well and grown in serum- and phenol red-free media.
• Purified mammamodulin prepared as described in Example 3B), was used at concentrations of 20 U/ml.
• IGF-I insulin-like growth factor type I
• E2 estradiol
• TNF-alpha tumor necrosis factor -alpha
• IL- 1 Interleukin-l ⁇
• the O.D. at 590 nm of the solubilized dye (crystal violet) which was taken up by the fixed cells during staining correlated linearly with cell numbers.
• the purified mammamodulin stimulated the proliferation of hormone- dependent cell line T47-D as shown in Fig. 12. In this figure, bars represent means of quadruplicates and standard deviations. IGF-I at the concentration used stimulated the cells maximally and mammamodulin had no additional effect on cell proliferation. Estradiol alone had little effect on cell growth but mammamodulin stimulated these cells to grow faster.
• the inhibitory effects of TNF-alpha and E -1 on T47-D proliferation were, at least partially, reversed by simultaneous addition of mammamodulin.
• MCF-7 The growth of MCF-7 was strongly stimulated by 25 U/ml MM as shown in Fig. 17.
• Cells were also stimulated by estradiol (E2) which was used as a control for normal function of the cells.
• E2 estradiol
• Fig. 17 the increases in cell numbers of stimulated cultures are expressed in percent of data obtained from the control cultures. The values represent means and standard deviations of quadruplicates.
• MCF-7 cells showed an increase of 186%, ZR-751 cells of 243% and T47-D cells of 161% above control cells.
• IGF-I insulin-like growth factor
• IO *8 M insulin-like growth factor
• estradiol 3 xlO" 9 M
• MM-containing conditioned media from fast proliferating hormone-independent cell line MDA-MB-231 demonstrably suppressed estrogen receptor (ER) levels of hormone-dependent cells.
• ER estrogen receptor
• MCF-7 cells exposure to MM-containing media (30 units/ml) for 2 days reduced the number of ERs to about 55% +/- 10% (S.D. from 5 determinations), and in ZR-75-1 cells, the number of ERs was about 32% +/- 20% in comparison to untreated cells, as measured by a binding assay with intact cells using tritiated estrogen (17- ⁇ estradiol).
• MCF-7 cells (2.5 x IO 6 ) in serum- and phenol red-free cultures were stimulated with 20 units ml of highly purified MM (Example 2, MonoS fraction between g and h), or with 1 x 10" d M estradiol in the absence or presence of MM. The cells were harvested 5 hours after treatment. RNA was then extracted from the cells and purified for mRNA on pre-packed spun columns (Pharmacia). The mRNA concentrations were measured and then 2.5 ⁇ g of each extract was loaded and resolved on an agarose gel.
• ER mRNA was probed for ER message by hybridization with a human ER probe (Fig. 13).
• ER mRNA was expressed in MCF-7 control cells (Fig. 13, lane 1).
• IO *9 M estradiol stimulated ER mRNA expression (Fig. 13, lane 2).
• MM reduces the mRNA expression considerably in the absence (Fig. 13, lane 3) as well as in the presence of IO" 9 M estradiol (Fig. 13, lane 4).
• ER and progesterone receptor (PR) proteins were measured by extraction of stimulated MCF-7 cells and appUcation of enzyme immuno assays (EIA).
• EIA enzyme immuno assays
• MCF-7 ceUs were seeded into 12-weU cell culture plates (Costar) in the presence of 5 % FCS at a density of 150,000 cells per weU. The next day, the medium was replaced by serum- and phenol red-free medium. The foUowing day, ceUs in dupUcate wells were stimulated with MM (200 U/ml) or estradiol (1 x IO -9 ). After 6 and 48 h, the media were removed and the ceUs frozen at -70°C for at least 12 hours.
• the cells were extracted according to the method described by Madeddu et al. [Eur. J. Cancer Clin. Oncol. 24, 385-390 (1988)1 by adding 225 ⁇ l of extraction buffer composed of 500 mM KC1, 10 mM KH2PO4, 1.5 mM EDTA and 5 mM Na-molybdate. The pH was adjusted to 7.4 with IM KOH. Beta-mercaptoethanol was added freshly before use at a concentration of 0.01 %. The buffer was at 4° C. After 90 min, the extracts were centrifuged in 1 ml Eppendorf tubes for 5 min at 12,000 x g and 4°C.
• supematants were coUected and 100 ⁇ l of extract used for receptor determination.
• ER and PR levels were measured using EIA kits manufactured by Abbott Laboratories, North Chicago, IL, USA. The results (Fig. 15) showed that 6 h after stimulation with MM, the ER protein levels of MCF-7 ceUs were slightly and after 48 h strongly reduced in comparison to controls. PR levels were similar to controls at both time points when ceUs were stimulated with MM.
• MCF-7 ceUs were seeded into 24-weU Falcon culture plates (2 cm 2 /weU) at a density of 150,000 ceUs/weU in ceU culture medium containing 5 % fetal calf serum (FCS). After 24 hours, the medium was exchanged with medium containing 1 % FCS. The following day, the ceUs were incubated for two hours with serum-free medium before stimulation. CeUs were treated with MM concentrations of 20 and 200 U/ml. An unstimulated control was included.
• FCS fetal calf serum
• MM stimulated the tyrosine-specific phosphorylation of a membrane protein of MCF-7 ceUs with an apparent molecular mass of approximately 180-190 kDa when compared with molecular weight standards and with a phosphorylated EGF receptor reference from EGF-stimulated A431 (Fig. 16).
• the identity of the phosporylated protein(s) was not determined.
• the surface receptor numbers were determined with flow cytometry using a mouse monoclonal antibody directed against the ceU surface domain of the human EGF receptor (Ab-1, Oncogene Science, Manhasset, NY) or the cell surface domain of erbB2 and a second, fiuorescein isothiocyanate (FITC)- labeled goat anti-mouse antibody (Becton Dickinson, San Jose, CA) was appUed to stain the ceU-bound mouse anti EGF receptor or anti erbB2 antibodies. The ceUs were scraped from the dishes, washed in serum-free medium and counted using a micro ceU counter (Sysmex F-300, TOA Medical Electronics, Kobe, Japan).
• FITC fiuorescein isothiocyanate
• At least 90% of aU ceUs were staining with the fluorescent antibody, as was confirmed by determining the number of ceUs with increased fluorescence in a gated dot plot on the flow cytometer. Bound fluorescence was calculated by subtracting the fluorescence of ceUs incubated only with the second (FITC-labeled) antibody from the fluorescence obtained from samples incubated with the first antibodies (the anti receptor antibodies) and the FITC-labeled antibodies.
• T47-D ceUs were grown in serum and phenol red-free ceU culture medium supplemented with transferrin, low levels of insulin and 1 mg/ ml bovine serum albumin (Albumax I from Gibco). The seeding density was between 25,000 and 35,000 ceUs per cm 2 . Two days after seeding, the cells were used for MM activity testing. T47-D ceUs reacted to MM similar to ZR-75-1 ceUs (compare Fig. 11a) but were sUghtly more sensitive and easier to handle because their attachment to the substrate is tighter.
• MM containing solutions were added in appropriate amounts to weUs containing the test ceUs.
• the culture plates were then immediately returned to the ceU culture incubator to maintain the normal 37°C temperature for ceU cultures. After 10 to 15 minutes, and a second time after 30-45 minutes, the ceUs were inspected by phase contrast microscopy at a magnification of 200 x for induced membrane ruffling and lameUipodia formation.
• MM is antigenic in npnhuman mammals, so that antibody is produced by the animals after they have been inoculated with the MM. Two doses of 5 ⁇ g MM are sufficient to induce production of antibody in mice. Polyclonal antisera can be isolated from the blood of the inoculated animals.
• Monoclonal antibody to MM is produced by conventional K ⁇ hler-MUstein processes: immunization of a suitable animal species by injection with MM, recovery of antibody-producing ceUs (i.e., spleen cells) sensitized to MM, immortalization of the antibody-producing ceUs by fusion with a compatible myeloma ceU line, and isolation of the monoclonal antibody from a selected immortalized ceU line thus established.
• ceUs i.e., spleen cells
• immortalization of the antibody-producing ceUs by fusion with a compatible myeloma ceU line
• isolation of the monoclonal antibody from a selected immortalized ceU line thus established Preferably, female Balb/c mice are injected with MM in Titremax (CytRx, 150 Technology Parkway, Technology Park Atlanta, Norcross, Georgia 30092), administered by i.p. injection. After two weeks, this injection is repeated.
• mice which exhibit the highest antibody levels are sacrificed, and their spleen ceUs are immortalized by fusion with mouse (Balb/c) myeloma ceUs, e.g., using PEG 4000, and distributed into 24 X 24 weUs.
• the hybridoma lines thus produced are screened and selected for the production of antibody.
• MM cDNA may be isolated from a random-primed cDNA library created using poly(A) + RNA from MDA-MB-231 ceUs.
• a vector e.g., Lambda ZAP II vector, is utilized to form the MM-fusion protein, and the expressed protein is then screened using MM antibody.
• the cDNA library may be screened using MM amino acid sequence information, e.g., by colony hybridization techniques, for example expressing the Ubrary in an expression system, preferably E.
• coU lysing the colonies, e.g., on nitroceUulose filters, denaturing their DNA in situ and fixing it on the filter, hybridizing with labeled, preferably radiolabeled, oligonucleotide probes of at least 24 base pairs having cDNA base sequences corresponding to aU or a portion of the amino acid sequence of MM, identifying hybridized colonies, and retrieving the corresponding vectors from the Ubrary, using chromosome walking techniques if necessary to isolate and characterize the entire cDNA.
• a suitable expression system e.g., a prokaryotic system such as E. coU.
• the MM may be isolated from the culture medium of the expression system, e.g., using the procedures outlined above. Trample 9
• Heparin is useful in the treatment or control of breast cancer due to its inhibitory effect on MM activity.
• Heparin for this use is preferably administered parenteraUy, most preferably by means of an implantable pump permitting long term, continuous intravenous infusion.
• heparin is relatively nontoxic, a trial dose of 1000 units should precede usual therapeutic dosages to confirm that the patient wiU not have an aUergic reaction.
• Heparin preparations should be used which are designed for long- term administration, e.g., have low anticoagulant and angiogenic activity.
• Monoclonal antibodies to MM may be used therapeuticaUy to block MM activity, thereby controlling or reducing the metastasis of breast cancer.
• Assay kits utilizing polyclonal or monoclonal antibodies (hereinafter collectively referred to as antibodies) to MM may be comprised of the following components: (i) antibody, preferably lyophilised; (ii) labeled (preferably radiolabeled) MM or fragment thereof; and (iii) MM standard containing a known amount of MM. If radiolabeled MM is used, it is preferably 125 I- labeled MM, labeled to about 50-100 ⁇ Ci/ ⁇ g.
• the antibody is dissolved and incubated together with the labeled MM or fragment and either the sample to be assayed or the MM standard. Incubation takes place preferably at cool temperatures, e.g., about 4°C, and lasts for at least two hours, preferably 4-6 hours.
• the pH of the incubating mixture is preferably kept in the range of from 5 to 8, more preferably at 7 or 8, preferably with the aid of a buffering agent such as a citrate or tris buffer.
• the fraction of labeled MM or fragment is separated from the unbound fragment, e.g., by the use of charcoal such as dextran-coated charcoal. The unbound fraction adsorbs to the charcoal and may then be separated by filtration or by centrifugation.
• the amount of radioactivity in one fraction is then measured by standard techniques, e.g., by Uquid scintillation counting after the addition of a secondary solute.
• the proportion of labeled MM or fraction bound to the antibody is inversely proportional to the amount of MM in the unknown plasma sample.
• a caUbration curve may be prepared by analyzing solutions of MM of known concentration.
• Nude mice inoculated with human mammary tumor cells are a preferred animal model for study of MM action and effects of blocking MM action by antagonists.
• nude mice are inoculated with hormone- dependent mammary tumor ceUs, e.g., MCF-7 ceUs, and the developing tumors are treated with MM (infusion) in the absence and presence of MM blockers such as heparin, neutraUzing MAbs, and MM receptor blockers, to evaluate the actions of MM in vivo on responder ceUs.
• hormone- dependent mammary tumor ceUs e.g., MCF-7 ceUs
• MM blockers such as heparin, neutraUzing MAbs, and MM receptor blockers
• nude mice are inoculated with hormone-independent mammary tumor ceUs, e.g., MDA-MB-231 ceUs, and the developing tumors are treated with heparin, blocking MAbs for MM, and MM receptor blockers to evaluate the role of MM in producer cell proliferation in vivo.
• hormone-independent mammary tumor ceUs e.g., MDA-MB-231 ceUs
• Results are based on the observation of morphological changes after stimulation of the mammamoduUn responder cells ZR-75-1 and T-47D by conditioned media (CM) of the ceUs listed. Fresh serum-free medium was added to confluent ceU cultures of each ceU (grown serum-free) and coUected 3 days later for the testing. The tests were performed as described in Example 6. "+” stands for unequivocaUy detectable ceU activation which is inhibited by 100 ⁇ g/ml heparin; (+) stands for marginal ceU activation.
• Hs578Bst, MDA-MB-453 and BT-20 are estrogen receptor-negative cells that have low growth rates in ceU culture and produce only very low or undetectable amounts of mammamodulin activity.

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• 1994
  • 1994-06-17 AU AU71858/94A patent/AU7185894A/en not_active Abandoned
  • 1994-06-17 JP JP7503781A patent/JPH08501456A/ja active Pending
  • 1994-06-17 NZ NZ268592A patent/NZ268592A/en unknown
  • 1994-06-17 EP EP94920949A patent/EP0659212A1/en not_active Withdrawn
  • 1994-06-17 WO PCT/EP1994/001975 patent/WO1995002052A1/en not_active Application Discontinuation
  • 1994-06-17 RU RU95116901/13A patent/RU95116901A/ru unknown
  • 1994-06-17 CN CN94190470A patent/CN1111910A/zh active Pending
  • 1994-06-17 CA CA002141417A patent/CA2141417A1/en not_active Abandoned
  • 1994-06-29 ZA ZA944687A patent/ZA944687B/xx unknown

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