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• • 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
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3038—Kidney, bladder
• • 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
  • 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
• • 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
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3015—Breast
• • 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
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3053—Skin, nerves, brain
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4703—Regulators; Modulating activity
  • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity

Definitions

• the present invention is related generally to the field of cancer diagnosis and management. More particularly, the present invention is related to novel tumor motility factors and their utility in devising new approaches to cancer diagnosis, prevention and therapy.
• an object of the present invention to identify and provide an autocrine factor controlling motility of tumor cells, such autocrine factor being designated herein as "AMF.” It is a further object of the present invention to provide antibodies having specific binding affinity for AMF or AMF receptors. It is a still further object of the present invention to provide a kit for detecting, localizing and predicting metastases and tumor angiogenesis in humans. It is yet another object of the present invention to provide a method of predicting, preventing and/or treating metastatic invasion and cancer proliferation in humans. It is an additional object of the present invention to provide a pharmaceutical composition comprising an effective amount of neutralizing antibodies against AMF to inhibit motility of tumor cells in a pharmaceutically acceptable carrier.
• Fig. 1 shows a schematic representation of the Boyden test
• Fig. 2 shows (a) Scatchard analysis of 125 I-AMF binding to suspended tumor cells; and (b) dose response curve of cell motility to purified AMF.
• polypeptide having the following properties: (a) secreted by mammalian cells and stimulates random locomotion of the producer cells; (b) having molecular weight of > 30,000; and (c) being inhibited by pertussis toxin.
• the polypeptide of the present invention is found to have, at least in part or in whole, the following amino acid sequence at its NH2 terminus (single letter code) or at the NH2 terminus of an active fragment of the polypeptide :
• MDA231 and MDA435 breast carcinoma cells lines were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. Both of these estrogen independent cell lines produce metastases in the lungs of a 6 week-old NIH nude mice, 6 weeks following injection of 5 x 10*5 cells into the lateral tail vein.
• DMEM Dulbecco's modified Eagle's medium
• Elution with PBS yields an active fraction that corresponds to material with a molecular weightof about 54 kDa.
• This fraction is dialyzed and concentrated 25 fold.
• the material is made up to 50 mM Tris-acetate, pH 8.0 and applied to a mono Q anion exchange column (source) and eluted with a linear salt gradient (0-1 M NaCl) with the following modification: When the NaCl concentration reaches 0.25 M, this concentration is held for 10 min before resuming the gradient.
• AMF is eluted in the 0.3 M to 0.4 M NaCl fraction.
• the active fraction is dialyzed and concentrated to a small volume (about 0.5 ml) which in turn is made up to 0.02 M phosphate in normal saline, pH 7.4.
• the assay of motility is accomplished by the use of a modified Boyden (Zigmond, et al, J. Exp. Med. 137:387-410, 1973) chamber.
• This is a device ( Figure 1) consisting of 2 wells horizontally separated by a microporous polycarbonate filter with a pore diameter of about 8 u.
• the motility stimulus (or chemoattractan ) is placed in the lower well to contact the filter.
• To the upper well is added a -suspension of cells (for example A2085 melanoma cells) at a concentration of about 10 ⁇ cells/ml.
• the chamber is then placed in a humidified incubator for about 4 hours at 37 degrees C in an atmosphere of air and about 5% CO2. During this time, the cells are deposited by gravity on the topside of the filter. However, some cells (about 5 to 10%) migrate to the underside of the filter in response to the motility stimulant. Expenditure of energy must occur during migration since the average diameter of the cell is greater than the pore size diameter.
• the filter is removed and subjected to a fixing and staining procedure. This includes first immersing the filters in a methanol-containing solution for about 2 minutes; then in an eosin solution for about 2 minutes; and then in a hematoxylin solution for about 3 minutes.
• the filters are washed in water and placed on a glass slide with the topside up.
• the buttons of stained cells on the topside are completely removed with a small piece of dry tissue paper.
• the stained cells that have migrated through the filter then become apparent. These are counted with the aid of a microscope at a magnification of about 500X.
• Five different high power fields are visualized with a grid in one ocular, the cells in 5 fields are counted and the average is computed.
• a ratio of _5 for positive control/negative control is indicative of a positive response of the cells to the motility stimulus.
• Random and Directed Motility Measurement of random motility is accomplished by exposing the cells to a fixed concentration of stimulus and determining their migration as described above. This includes adding equal increasing concentrations of attractant to both upper and lower wells prior to the assay incubation. The random migration of cells as a function of the levels of attractant is then determined. Directed migration occurs if the cells migrate better in - 7 -
• DMEM supplements with L-glutamine (2 ⁇ g), penicillin and streptomycin with or without 10% heat-inactivated fetal calf serum were purchased from commercial sources such as Meloy Laboratories, Inc. (Springfield, VA). Pertussis toxin and cholera toxin were obtained from List Biological Laboratories, Inc. (Cambell, CA).
• Phorbol 12-myristate 13-acetate (PMA), phorbol 12, 13-didecanoate (PDD), calcium ionophore A23187, diltiazem, nifedipine, verapamil, trifluoperazine, leupeptin, forskolin and 8-Br cAMP were all purchased from Sigma Chemical Company (St. Louis, MO).
• the l-oleoyl-2-acetylglycerol was from Molecular Probes (Eugene, OR).
• the Nucleopore membranes (polyvinyl- pyrrolidone-free) as well as the 48-well chemotaxis chamber were purchased from Neuro Probe, Inc. (Cabin- John, MD).
• A2058 melanoma cells (approximately 75-90% confluent) were .harvested with trypsin-EDTA and allowed to recover at room temperature in DMEM supplemented with 10% fetal calf serum for at least one hour. The cells were then resusoended at 2 x 10 ⁇ /ml in DMEM with 1 mg/ml bovine serum albumin. The assay was performed in 48-well micro-chemotaxis chamber (Harvath et al, 1980 supra) with 8 ⁇ m Nucleopore membranes coated with type IV collagen. The chambers were incubated at 37 degrees C for 4-5 hours, then developed using Diff Quick stains (American Scientific).
• the stained membranes were placed onto glass slides with the original cell side up so that the cell pellet could be wiped from the surface. Cells that had migrated through the pores were trapped between glass and membrane and could be easily counted by light microscopy under high power field (500X). Unstimulated random migration was ⁇ 20% of directed migration.
• chemicals Prior to or during the chemotaxis assay, chemicals could be co-incubated with cells to alter cellular metabolism or stimulate a chemokinetic response. At the start of the assay, chemicals could also be added to the lower chamber to demonstrate chemotactic potential.
• AMF protein (10 ⁇ g) was emulsified with complete Freund's adjuvant and injected into the foot pad of 3 C3H mice. Two weeks later the mice were boosted with 5 ug of AMF in PBS injected intravenously in the tail vein in a volume of 0.1 ml. One month later the mice were bled and the serum was tested for its ability to inhibit tumor cell motility. In this assay the mouse sera was preincubated with the AMF in the Boyden chamber migration assay. At a dilution of 1/1000 the mouse sera produced 90% inhibition of tumor cell motility compared to pooled mouse sera control.
• Purified AMF protein (10 ⁇ g) was emulsified in complete Freundi ' s adjuvant and injected- into a subcutaneous site on the back of New Zealand white rabbits. Booster injections of 5 ⁇ g were applied at 6 and 12 weeks. At 3 and 4 months the rabbits were bled and the sera was tested for motility inhibition activity. At a dose of 1/1000 the immune sera abolished motility compared to control preimmune sera. The sera were heat inactivated at 56°C for 30 minutes.
• the purity of the isolated AMF was determined by the following criteria: (a) Single 54 kDA band was found on a single and two dimensional polyacrylamide gel electrophoresis performed by standard procedures well known in the art. Protein was identified with silver stain. (b) Protein band cut from the gel retains motility stimulating activity. (c) NH2 terminus amino acid sequence (1-19) reveals one "type of amino acid residue at each cycle; and (d) Murine and rabbit anti-AMF antibodies block the motility stimulating activity of human tumor AMF. Based on the above criteria, the isolated AMF of the present invention was found to be substantially pure. The term "substantially" as used herein means as pure as it is possible to obtain by standard techniques.
• Cholera Toxin in contrast to pertussis toxin, is thought to act on the G s protein that stimualtes adenylate cyclase to produce the second messenger, cAMP. Cholera toxin was added to A2058 cells either for overnight incubation in flasks or for variable periods of preincubation prior to the start of the chemotaxis assay. The tested doses of cholera toxin ranged from about 0.1-50 ⁇ g/ml.
• Cholera toxin is thought to act by ADP-ribosylation of the G s protein in an active configuration that can stimulate adenylate cyclase. Since the effect of cholera toxin on A2058 cell motility was minimal, further tests were conducted to determine whether other agents that act on the cAMP pathyway would be inhibitory. Forskolin stimulates adenylate cyclase directly without acting through an intermediary G protein. The cAMP analogue, 8-Br cAMP, is able to enter intact cells.
• the migration-stimulating material of the present invention is found to have a molecular weight of about 54 kilodaltons. This form may be a precursor of an active factor. It is possible that cellular or serum components could activate or inhibit the action of the motility factor.
• the motility factor is inactivated by exposure to streptococcal protease, but active chymotrypsin-derived fragments can be produced (data not shown). The activity is destroyed by boiling but- is stable upon exposure to 56 degrees C.
• the activity is stable to a pH range from 4 to 11 (data not shown).
• AMF autocrine material
• known growth factors such as PDGF, c ⁇ TGF, ⁇ TGF, EGF, IGF, transferrin, or FGF do not substitute or block the AMF (data not shown).
• Amino acid analysis indicated a unique sequence of 19 amino terminal amino acids of AMF.
• a slightly small form of the active material was also found to have a unique amino terminal sequence. Protein data base searches failed to reveal any other polypeptide with such a sequence. It has also been found that motility induction by AMF is not blocked or substituted by known growth factors or serum factors.
• AMF markedly stimulates the random and directed motility of breast cancer cells but fails to induce motility in leukocytes.
• the factor also stimulated random pseudopodia production by breast carcinoma cells and melanoma cells.
• AMF and its receptor are enhanced more than 100 fold in certain cells.
• Antibodies recognizing AMF abolish human tumor cell motility in vitro without altering tumor cell viability.
• the availability of an isolated and purified autocrine, polypeptide, tumor motility factor makes it possible to obtain anti-AMF antibodies having specific binding affinity for said motility factor.
• Such antibodies can either be polyclonal or monoclonal and are prepared by well known standard techniques routine in the art. Such antibodies can also be labelled with suitable radioisotopes or fluorescent and other markers or ligands and employed for the detection, quantitation and/or localization of the AMF in human tissue or body fluid. Furthermore, radiolabelled AMF together with unlabelled AMF can be utilized in a standard competitive assay to measure AMF receptor level. Such binding assay for determining the receptor level is carried out as follows. AMF Binding Assay: Purified AMF is iodinated using the standard " Bio Rad enzymobead procedure.
• AMF binding exhibits saturation with 80% specific * binding and about 30,000 receptors per cell.
• Scatchard analysis according to standard methods shows a linear relationship between the specifically bound/free ratio and the specifically bound .AMF, with an estimated kd in the range of about 0.5 nM. Detection of cancer in humans is also made possible by the present discovery and testing of human body samples for this purpose is now illustrated using urine samples from bladder cancer patients.
• Urine samples from patients with bladder cancer are collected and processed with centrifugal microconcentrator (AMICON) with an exclusion filter of 10 kilodaltons.
• the processed urines are reconstituted at a 10-fold concentration with steriel phosphate buffered saline pH7.5 and stored at -20°C until use.
• Tumor grade is determined by a pathologist using a scale of one to three with grade one tumors showing the most differentiation and grade three tumors showing the least differentiation.
• Bladder tumors are staged according to ' , the American Joint Committee TNM classification.
• the preferred cell line is human MDA 435 cells (ATCC).
• ATCC human MDA 435 cells
• the concentrated urine samples are applied to the microwell migration chamber assay as described herein supra. Each sample is tested at a series of dilutions with and without the addition of the antibodies directed against human tumor AMF.
• AMF units are recorded as the proportion of tumor cells stimulated to migrate by the sample which is inhibited by the antibodies. In general, greater than 80% of the stimulated migration is inhibited by an antibody concentration of about 10 ⁇ g/ml.
• control urines with non-neoplastic disorders such as kidney stones failed to contain significant levels of motility factors. All of the bladder transitional cell carcinoma cases exhibited a positive motility response in the urine. The highest levels of motility factor production was found in the urine of patients with high grade tumors or with stage D (metastatic) tumors.
• TCC Transitional cell carcinoma of the bladder
• TCC stg D metastatic TCC
• the antibodies against AMF can be employed to block or inhibit AMF activity thereby arresting tumor invasion or metastatic proliferation which depend on tumor cell motility.
• Availability of such neutralizing antibodies also makes it possible to treat such conditions as breast carcinoma and melanoma by administering to a person inflicted with these conditions, an effective amount of the AMF-antibodies to prevent these conditions from progressing.
• a pharmaceutical composition for treating cancer and metastases is prepared by simply including an effective amount of neutralizing antibodies against AMF to inhibit motility of tumor cells and a pharmaceutically acceptable carrier such as physiological saline, non-toxic buffers and the like.
• kits for detecting tumor aggressiveness and/or metastatic activity comprising separate containers containing (a) antibodies having specific binding affinity for AMF; (b) labelled AMF; (c) unlabelled AMF and instructional material for performing tests utilizing the antibodies and the AMF provided in the kit for determining AMF and/or receptor activity in a body sample.
• Such accessories as microtiter plates, micropipettes, means for reading antibody titer and the like are routinely found in such kits and may be included for convenience in the kits of the present invention.
• the present invention provides a new tool for understanding mechanisms which control tumor cell invasion and opens new strategies for cancer diagnosis and therapy. Epithelial cells do not normally exhibit invasive behavior.
• the motility factor described herein does not affect the migration of normal blood leukocytes. Therefore, a therapeutic agent aimed at inhibiting the factor described in the invention should have low toxicity against normal resting tissues.
• Pharmacologic preparations obtained in accordance with the present invention which inhibit invasion of tumor cells and prevent the transition from in situ to invasive carcinoma could be potent cancer arresting agents. Inhibitors of tumor invasion can also prevent the growth of established metastases because a metastasis may need to invade locally as it grows. Furthermore, such agents may inhibit tumor angiogenesis.
• Antibodies to motility factors or their receptors could be applied through tissue immunohistology, radioscintography, or serum immunoassays to localize metastases and predict cancer aggresseiveness in individual patients.
• autocrine motility factors or their receptors define a new class of oncogenes.
• the level of expression of these genes in a patient's tumor may provide important diagnostic information through monitoring the level of AMF in the body sample.
• invasion and metastases are among the major causes of cancer treatment failure.
• the present invention provides new clinical strategies to (a) detect pre-invasive lesions and prevent their progression; (b) accurately predict the aggressiveness of a patient's tumor, and (c) identify and eradicate micrometastases.
• One of the least understood aspects of tumor invasion is tumor cell locomotion.
• the present invention allows the determination of the role of the tumor cell motility factor.

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• 1988
  • 1988-05-27 AU AU18034/88A patent/AU614755B2/en not_active Ceased
  • 1988-05-27 JP JP63505028A patent/JP2851288B2/ja not_active Expired - Fee Related
  • 1988-05-27 EP EP19880905318 patent/EP0362278A4/de not_active Withdrawn
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