AU614755B2 - Autocrine motility factors in cancer diagnosis and management - Google Patents

Autocrine motility factors in cancer diagnosis and management Download PDF

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AU614755B2
AU614755B2 AU18034/88A AU1803488A AU614755B2 AU 614755 B2 AU614755 B2 AU 614755B2 AU 18034/88 A AU18034/88 A AU 18034/88A AU 1803488 A AU1803488 A AU 1803488A AU 614755 B2 AU614755 B2 AU 614755B2
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amf
motility
cells
cancer
antibodies
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Lance A. Liotta
Elliott Schiffmann
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [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
    • C07K16/3038Kidney, bladder
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702Regulators; Modulating activity
    • C07K14/4705Regulators; Modulating activity stimulating, promoting or activating activity
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [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
    • C07K16/3015Breast
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [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
    • C07K16/3053Skin, nerves, brain
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701Details
    • G01N2333/4703Regulators; Modulating activity
    • G01N2333/4706Regulators; Modulating activity stimulating, promoting or activating activity

Description

w (0~o

PCT

WVORLD INTELLECTUAL PROPERTY ORGANIZATION Internuainal Bureau AU-AI1-18034/88 INTERNATIONAL APPLICATION PUBLISHED UDER THE PATENT COOPERATION TREATY (PCT) (21) International Application Number: PCT/US88/01805 (74) Agents: STERN, Marvin, R. et Holman Stern, 2401 Fifteenth Street, Washington, DC 20009f (22) International Filing Date: 27 May 1988 (27.05.88) (US).

(31) Priority Appllcatkin Number: 058,381 (81) Designated States: AT (European patent), AU, BE (Eu-' ropean patent), CH (European patent), DE (Euro-' (32) Priority Date: 5 June 1987 (05,06,87) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European pa- (33) Priority Country: us tent), NL (European patent), SE (European, patent), (71) Applicant: THE UNITED STATES OF AMERICA, as Publiished represented by THE SECRETARY, U.S. DE~PART- Wit/h international search report.

MENT OF COMMERCE 5285 Port RoyalA.* AR18 Road, Springfield, VA 22161

A

(72) Inventors; LIOTTA, Lance, A. ,9207 Mistwood Drive, Potomac, MD 20854 SCM IFFMANN, Elliott :AUSTRALIAN 3207 Pickwick Lane, Chevy Chase, MD 20815 AN18 EA

J

PATENT OFFICE (54)Title: AUTOCRINE MOTILITY FACTORS IN CANCER DIAGNOSIS AND MANAGEMENT (57) Abstract The present invention describes an isolated and substantially pure mammaiian cell polypeptide which stimulates random locomotion of' producer cell and wtloh has a molecular weight greater than 30,000. The unique polypeptide of the present invention is inhibited by pertussis toxin, A kii and method for detecting metastasis in human are also described.

S WO 88/09797 PCT/US88/01805 1 AUTOCRINE MOTILITY FACTORS IN CANCER 2 DIAGNOSIS AND MANAGEMENT 3 BACKGROUND OF THE INVENTION 4 Technical Field The present invention is related generally to the 6 field of cancer diagnosis and management. More 7 particularly, the present invention is related to novel 8 tumor motility factors and their utility in devising new 9 approaches to cancer diagnosis, prevention and therapy.

State of the Art 11 Cell motility is necessary for tumor cells to 12 traverse many stages in the comnlex cascade of invasion 13 and metastases. Such stages include the detachment and 14 subsequent infiltration of cells from the primary tumor into adjacent tissue, the migration of the cells through 16 the vascular wall into the circulation (intravasation), 17 and extravasation of the cells to a secondary site. The 18 movement of cells through biological barriers such as the 19 endothelial basement membrane of the vasculature may occur by means of chemotactic mechanisms. Studies on in S 21 vitro chemotaxis of some tumor cells indicate that a 22 variety of compounds such as complement-derived 23 materials, collagen peptides, formyl peptides, and 24 certain connective tissue components can act as WO 88109797 PCTUS88/'180o -2- 1 chemoattractants. Todaro, et al. (Proc. Natl. Acad. Sci 2 USA, 77:5258-5262, 1980) reported autocrine growth 3 factors for transformed cells. Other growth factors of 4 various types are also known. However, the existence and role of an autocrine factor controlling chemotactic 6 (directional) and chemokinetic (random) motility of tumor 7 cells has not heretofore been known or described. It may 8 be important to note here that cell motility is an aspect 9 of cell behavior distinct from cell growth and proliferation.

11 SUMMARY OF THE INVENTION 12 It is, therefore, an object of the present invention 13 to identify and provide an autocrine factQr controlling 14 motility of tumor cells, such autocrinu factor being designated herein as "AMF." 16 It is a further object of the present invention to 17 provide antibodies having specific binding affinity for 18 AMF or AMF receptors.

19 It is a still further object of the present invention to provide a kit for detecting, loc-lizing and 21 predicting metastases and tumor angiogenesis in humans.

22 It is yet another object of the present invention to 23 provide a method of predicting, preventing and/or 24 treating metastatic invasion and cancer proliferation in humans.

26 It is an additional object of the present invention 27 to provide a pharmaceutical composition comprising an 28 effective amount of neutralizing antibodies against AMF 29 to inhibit motility of tumor Cells in a pharmaceutically acceptable carrier.

I wp V O 88/09797 PCT/US88/O1805 3 1 Various other objects and advantages of the present 2 invention will become evident from the Detailed 3 Description of the Invention.

4 BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many of the 6 attendant advantages of the invention will be better 7 understood upon a reading of the following detailed 8 description when considered in connection with the 9 accompanying drawings wherein: Fig. 1 shows a schematic representation of the 11 Boyden test; and 12 Fig. 2 shows Scatchard analysis of 125 1-AMF 13 binding to suspended tumor cells; and dose response 14 curve of cell motility to purified AMF.

DETAILED DESCRIPTION OF THE INVENTION 16 The above and various other objects and advantages 17 of the present invention are achieved by a polypeptide 18 having the following properties: secreted by 19 mammalian cells and stimulates random locomotion of the producer cells; having molecular weight of 30,000; 21 and being inhibited by pertussis toxin. The 22 polypeptide of the present invention is found to have, at 23 least in part or in whole, the following amino acid 24 sequence at its NH 2 terminus (single letter code) or at the NH 2 terminus of an active fragment of the 26 polypeptide: 27 D K ELRFRDCTKSLAEAN K

-J

FAr WO 88/09797 PCTIUS88/01805 I

I

4 1 Unless defined otherwise, all technical and 2 scientific terms used herein have the same meaning as 3 commonly understood by one of ordinary skill in the art 4 to which this invention belongs. Although any methods and materials similar or equivalent to those described 6 herein can be used in the practice or testing of the 7 present invention, the preferred methods and materials 8 are now described. All publications mentioned hereunder 9 are incorporated herein by reference.

MATERIALS AND METHODS 11 Cell Lines 12 Human MDA231 and MDA435 breast carcinoma cells lines 13 were obtained from ATCC and cultured in Duloecco's 14 modified Eagle's medium (DMEM) supplemented with fetal bovine serum. Both of these estrogen independent 16 cell lines produce metastases in the lungs of a 6 17 week-old NIH nude mice, 6 weeks following injection of 38 5 x 105 cells into the lateral tail vein.

19 Isolation and Purification of the Autocrine Motility Factor 21 MDA231 and MDA435 human breast carcinoma cells are 22 grown in DMEM to 60% confluency in the absence of added 23 protein. The media is lyophilized and the residue 24 dissolved in about 2 ml of distilled H20. This solution is applied to a P'D-10 (Sophadex G25 medium) column. The 26 first 2.5 ml are discarded and the next 4 ml are 27 collected. The effluent contains AMF separated from low 28 molecular weight material. This collected fraction is 29 made up to 0.02 M phosphate buffered saline, pH 7.4 (PBS) with 10 x PBS and applied to a Sephacryl S-300 column n 31 PBS (source of column). Elution with PBS I I IWO 88/09797 PCT/US88/O1S05 5 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 2 9 31 32 33 yields an active fraction that corresponds to material with a molecular weighY'of 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. This is applied to a heparin column in PBS. The column is eluted with a linear gradient of NaCl (0.15 M to 1 M) which elutes AMF between 0.35 M and 0.4 salt gradient. After each purification step, column fractions (dialyzed to remove salt) are assayed for motility stimulating activity by the modified Boyden chamber procedure.

Assay Procedure for Cell Motility 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 chemoattractant) is placed in the lower well to contact the filter. To the upper well is added a zuspension of cells (for example A2085 melanoma cells) at a concentration of about 106 celis/ml. The chamber is then placed in a humidified incubator for about 4 hours at 37 dogrees C in an atmosphere of air and about St CO 2 During this time, the cells are deposited by gravity on the topside .4 WO 88/09797 -6 1 of the filter. However, some cells (about 5 to 2 migrate to the underside of the filter in response to the 3 motility stimulant. Expenditure of energy must occur 4 during migration since the average diameter of the cell is greater than the pore size diameter. At the end of 6 the incubation pericd, the filter is removed and 7 subjected to a fi,xing and staining procedure. This 8 includes first immersing the filters in a 9 methanol-containing~ solution for about 2 minutes; then in an eosin solution for about 2 minutes; and then in a 11 hematoxylin solution for about 3 minutes. Thereafter the 12 filters are washed in water and placed on a glass slide 13 with the topside up. V~ie buttons of stained cells on the 14 topside are completely removed with a small piece of dry tissue paper. The stained cells that have migrated 16 through the filter then become apparent. These are{ 17 counted with the aid of a microscope at a magnification 18 of about 500X. Five different high power fields are 19 visualized with a grid in one ocuiar, the cells in fields are counted and the average is computed. A ratio 21 of >5 for positive control/negative control is indicative 22 of a positive response of the cells to the motility 23 stimulus.

24 Determination of Random and Directed (Chemotactic) Motility1* 26 Measurement og random motility is accomplished by 27 exposing the cells to a fixed concentra~tion of stimulus 28 and determining their mi.gration as described above. This 29 includes adding equal increasin-g concentrations of attractant to both upper and lower wells prior to the 31 assay incubation. The random mi.gration of colls as a 32 function of the levels of at'tract'ant is then determined.

33 Directed migration occurs if the cells migrate better in tYI-,. 3

I

WO 88/09797 PCT/US88/01805 7 positive gradients (higher concentrations of attractant in the lower well compared to the upper well) than in negative gradients (higher concentrations in upper well than in lower well). The results of such an assay are shown in the "checkerboard" tabulation of the results (Table It can be seen that random motility is quite significant for the A2058 melanoma cells responding to the AMF.

TABLE 1 Motility Factor in Upper Well Motility Factor In Lower Well 100 244 512 494 494 1056 825 1469 1781 1550 2144 2640 2800 2550 2262 4362 'diagonal' shows random migration triangle shows directed migration of gradient of motility stimulus.

of cells.

cells in a Lower positive i-r WO 88/09797 PCT/US88/01805.

8 1 Assay for Cell Pathways Involved in AMF Induced Motility 2 Materials: DMEM supplements with L-glutamine (2 pg), 3 penicillin and streptomycin with or without 4 heat-inactivated fetal calf serum were purchased from commercial sources such as Meloy Laboratories, Inc.

6 (Springfield, VA). Pertussis toxin and cholera toxin 7 were obtained from List Biological Laboratories, Inc.

8 (Canbell, CA). Phorbol 12-myristate 13-acetate (PMA), 9 phorbol 12, 13-didecanoate (PDD), calcium ionophore A23187, diltiazem, nifedipine, verapamil, 11 trifluoperazine, leupeptin, forskolin and 8-Br cAMP were 12 all purchased from Sigma Chemical Company (St. Louis, 13 MO). The l-oleoyl-2-acetylglycerol was from Molecular 14 Probes (Eugene, OR). The Nucleopore membranes (polyvinyl- pyrrolidone-free) as well as the 48-well 16 chemotaxis chamber were purchased from Neuro Probe, Inc.

17 (Cabin John, MD).

18 Cell Culture: The human melanoma cell line A2058 19 was maintained as described by Todaro et al, supra.

Production of Autocrine Motility Factor: In a 21 modification of the previously described technique 22 (Liotta et al, Proc. Natl. Acad. Sci. USA 83:3302-3306, 23 1986), A2058 cells were innoculated for 48 hours in DMEM 24 without any protein supplement. The medium was concentrated using a Centricon ultrafiltration assembly, 26 molecular weight cut off 30,000 daltons.

27 Chemotaxis Assay: The assay used to determine cell 28 motility was a modification of the techniques described 29 by Harvath et al, 1980.... Liotta et al, 1986 supra. In accordance with this technique A2058 melanoma cells 31 (approximately 75-90% confluent) were harvested with 32 trypsin-EDTA and allowed to recover at room temperature 33 in DMEM supplemented with 10% fetal calf serum for at 34 least one hour. The cells were then resuspended at j, p. 1 NO 88/09797 PCT/US88/01805 9 1 2 x 10 6 /ml in DMEM with 1 mg/mi bovine serum albumin.

2 The assay was performed in 48-well micro-chemotaxis 3 chamber (Harvath et al, 1980 supra) with pm Nucleopore 4 membranes coated with type IV collagen. The chambers were incubated at 37 degrees C for 4-5 hours, then 6 developed using Diff Quick stains (American Scientific).

7 The stained membranes were placed onto glass slides with 8 the original cell side up so that the cell pellet could 9 be wiped from the surface. Cells that had migrated through the pores were trapped between glass and membrane 11 and could be easily counted by light microscopy under 12 high power field (500X). Unstimulated random migration ,3 was <20% of directed migration.

14 Prior to or during the chemotaxis assay, chemicals could be co-incubated with cells to alter cellular 16 metabolism or stimulate a chtmokinetic response. At the 17 start of the assay, chemicals could also be added to the 18 lower chamber to demonstrate chemotactic potential.

19 Production of Murine Antibodies to AMF Purified AMF protein (10 pg) was emulsified with 21 complete Freund's adjuvant and injected into the foot pad 22 of 3 C3H mice. Two weeks later the mice were boosted 23 with 5 ug of AMF in PBS injected intravenously in the 24 tail vein in a volume of 0.1 ml One month later the mice were bled and the serum was tested for its ability 26 to inhibit tumor cell motility. In this assay the mouse 27 sera was preincubated with the AMF in the Boyden chamber 28 migration assay. At a dilution of 1/1000 the mouse sera 29 produced 90% inhibition of tumor cell motility compared to pooled mouse sora control. Purified AMF protein 31 ug) was emulsified in complete Freundi's adjuvant and 32 injected into a subcutaneous site on the back o2 Now 33 Zealand white rabbits. Booster injections of 5 Vg were WO 88/09797 PCT/US8810180' 10 1 applied at 6 and 12 weeks. At 3 and 4 months the rabbits 2 were bled and the sera was tested for motility inhibition 3 activity. At a dose of 1/1000 the immune sera abolished 4 motility compared to control preimmune sera. The sera were heat inactivated at 56 0 C for 30 minutes.

6 Determination of AMF Purity 7 The purity of the isolated AMF was determined by the 8 following criteria: 9 Single 54 kDA band was found on a single and two dimensional polyacrylamide gel electrophoresis 11 performed by standard procedures well known in the 12 art. Protein was identified with silver stain.

13 Protein band cut from the gel retains motility 14 stimulating activity.

NH

2 terminus amino acid sequence (1-19) reveals one 16 type of amino acid residue az each cycle; and 17 Murine and rabbit anti-AMF antibodies block the 18 motility stimulating activity of human tumor AMF.

19 Based on the above criteria, the isolated AMF of the present invention was found to be substantially 21 pure. The term "substantially" as used herein means as 22 pure as it is possible to obtain by standard techniques.

23 Amino Acid Sequencing 24 Edman degradation of purified AMU is performed with the Applied Biosystems (Foster City, CA) model 470A 26 gas-phase sequencer using the trifluoracetic acid 27 chemistry provided by the manufac urer, The 28 phenylthiohydantoin amino acids were identified and 29 quantitated by using the Perking-Elmer series 3B HPLC and ultraviolet detection.

SO 88/09797 PCTi';S88101805 11 1 Dose Ro1-onse and Time Course of Pertussis Toxin and 2 Effect on Motility: Pertussis toxin (PT) was added to 3 A2058 for overnight culture in flasks, for various period 4 of preincubation prior to an assay, or at different times after the start of an assay. PT doses that were tested 6 ranged from about 10 ng/ml to 1.5 pg/ml. Cell viability 7 at any of the tested doses was comparable to the 8 viability in untreated control Treated and 9 untreated cells were then tested for their motility response to the A2058 conditioned medium. Cell motility 11 in response to the DMEM alone was included as a negative 12 control for each treatment group of cells.

13 Overnight incubation of the cells with any of the 14 tested PT doses resulted in significant inhibition of cell motility (Table Preincubation for 30 minutes to 16 2 hours at doses of 0.5 1.5 ug/ml also resulted in 17 greater than 50% inhibition. When pertussis toxin was 18 added at the start of the assay or later, there was a 19 gradual diminution in the inhibitory effect. By 1-2 hours after the start of the assay, PT had minimal effect 21 on the observed motility.

22 The dose response of PT was consistent with 23 previously described inhibitory doses of PT for Gi and G o 24 proteins. The time course showed much diminished inhibition when PT was added at inadequate doses or for 26 insufficient time to saturate the G protein sites.

27 Hence, the data obtained in the present testing was 28 consistent with the hypothesis that AMF stimulates cell 29 motility through a receptor which requires a G protein to activate the cells.

PcIu-s8/01805, WO 88/09797 12 TABLE 2 AMF TREATMENTDATA Treatment P'ase K DNAase 2 g/ml RNAase PMSF 5 mM DDT 10 mM Heating 1OOC Heating 56C pH 4,0 pH 7.4 pH 11.0 Motility C ofE Controls) 13.2 95.1 104 95.5 97.2 100 100 PERTUSSIS TOXIN INHIBITION OF AMF INDUCED MOTILITY Time Pertussis Toxin Added (hrs. from start of assay) Percent Inhibit =.r of AMF Induced Motilitv Start of Assay* 0 zoo 9S 62 0 (n i ifl hi t n, no inhibit-,; .vil* *Tia o4. additiot v-1F *4Prtussfl Toxin requires at 1040t I hou- to penetratx coll =mbrano: and inhibit G protains by AD? F 1 i -i WO 88/09797 PCT/US88/01805 13 1 2 3 4 16 7 18 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 29 31 32 Cholera Toxin Dose Response and Time Course: Cholera toxin (CT) in contrast to pertussis to.:in, is thought to act on the Gs protein that stimualtes adenylate cyclase to produce the second messenger, CAMP. Cholera to::in 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 pig/ml. At all tested doses, cell viability was comparable to that of untreated cells Treated and untreated cells were then tested for chemotactic response to A2058 conditioned medi.um.

Overnight treatment with CT caused a diminished response to the A2058 conditioned medium,though the inhibitory effect was never complete (30-60 inhibition). If the cells were xposed to cholera toxin for just a brief preincubation prior to the start of the chemotaxis assay, the inhibition was minimal Effect of Other Agents Involved in the Adenviate cvclase System on Cell Motility: Cholera toxir is thought to act by ADP-ribosylation of the Gs protein in an active configuration that can stimulate adenylate cyclaso.

Since the effect of cholera toxin on A205 cell motility was minimal, further tests wore conducted to determine whether other agents that act on the CAMP pathyway would be inhibitory. Forakolin stimulates adenylate cyclase directly without acting through an intermediary G protein, The cAMP analogue, 8-Br cAMP, is able to ont=O intact cells. Both chemiclsa .ero added to A2058 OallS either for overnight incubatior in I1saku or for a 2 hour preincubation prior to the start of' chemotaxi. Both exhibited only a nartial inhibition of cell motilty that ii-. 1 WO 88/09797 PCTIUS88/aI805 -14-

I

Ii 1 was essentially identical to that of cholera toxin for 2 comparable periods of time.

3 Since these cells respond in a dose-dependent manner 4 to various concentration of conditioned medium obtained by incubating confluent cells in serum-free medium, it 6 was concluded that the motility factor is derived from 7 the cell. Results obtained with the modified Boyden 8 chamber experiments also demonstrate that the autocrine 9 factor of the present invention has both chemotactic (directional) and chemokinetic (randomly motile) 11 properties. Since the random stimulation was found to be 12 about three-fold greater than the directed motility, it 13 was concluded that the cells respond to gradients of the 14 motility factor as we.,l as to high uniform concentrations of the attractant.

16 When determined by gel filtration and gel 17 electrophoresis, the migration-stimulating material of 18 the present invention is found to have a molecular weight 19 of about 54 kilodaltons. This form may be a precursor of an active factor. It is possible that ueilular or serum 11 components could activate or inhibit the action of the 22 motility factor. The motility factor is inactivated by 23 exposure to streptococcal protease, but active 24 chymotrypsin-dorived fragments can be produced (data not shown). The activity ie destroyed by boiling but is 26 stable upon exposure to 56 degrees C. Additionally, the 27 activity is stable to a pH range from 4 to 11 (data not 28 shown). These properties indicate that the autocrine 29 material (AMF) of the present invention is different from a variety of known growth factors and chemnattractants.

31 It was also found that known growth factors such as 32 PDGF, aTGF, 8TGF, EGF, IGF, transferrin, or FGF do not 33 substitute or block the AMP (data not shown). Amino acid i 34 analysis indicated a unique sequence of 19 amino terminal -4 or 1 W088/09797 PCT/US88/01805 15 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 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 block6d or substituted by known growth factors or serum factors. lit a concentration of 1 nM or less, 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.

N

Following trasfection with the activated ras-oncogene, and its receptor are enhanced more than 100 fold in certain cells. Human breast carcinoma cells, but not normal breast epithelium, produce large quantities of AMF. 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 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 iarried out as follows.

WO 88/09797 PCT/US88/01805" 16 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 26 27 28 29 31 32 33 AMF Binding Assay: Purified AMF is iodinated using the standard Bio Rad enzymobead procedure. Increasing amounts of labeled AMF is incubated in a volume of 1 ml with 100,000 A2058 melanoma cells, in the presence or absence of 100 fold excess cold competitor. Incubation is conducted at 37*C for 40 minutes and the cell-bound radioactivity is separated by centrifugation. 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 tollected and processed with centrifugal microconcentrator (AMICON) with an exclusion filter of kilodaltons. The processed urines are reconstituted at a 10-fold concentration with steriel phosphate buffered saline pH7.5 and stored at -20 0 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.

Assay of Urine Samples: Although any cell line which responds to AMF can be omployadt the preferred cell line is human MDA 435 cells (ATCC). The concentrated urine samples are appliad to the microwall migration chamber assay as described herein 7M *1.

PCT/US88/01805 W088/09797 1 2 3 4 6 8 9 11 12 13 14 17 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 pg/ml.

As shown- in Table 3, 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.

I

WO 88/09797 C/S8010 PCT/US88/01805- TABLE 3 Urine Sample Control ks 75 Control ks 76 Ca in Situ Papillary TCC TCC 77 TCC 6 9 TCC 73 Recur TCC 79 TCC 11 485 TCC 11 491 TCC 11 554 TOC 111 457 TCC stg D 584 AMP units 5 9 32 64 44 98 123 130 169 105 41 72 234

SE

2 8 3 14 32 22 14 8 12 6 TCC =Transitional cell carcinoma of Recur TCC Recurrent TCC TCC II grade II TCC III =grade III TCC stg D metastatic TCC KS Kidney stones 2Z w Standard error the bladder WO 88/09797 PCT/US88/01805 19 1 Of course, the antibodies against AMF can be 2 employed to block or inhibit AMF activity thereby 3 arresting tumor invasion or metastatic p:oliferation 4 which depend on tumor cell motility. Availability of such neutralizing antibodies also makes it possible to 6 treat such conditions as breast carcinoma and melanoma by 7 administering to a person inflicted with these a conditions, an effective amount of the AMF-antibodies to 9 prevent these conditions from progressing. A pharmaceutical composition for treating cancer and 11 metastases is prepared by simply including an effective 12 amount of neutralizing antibodies against AMF to inhibit 13 motility of tumor cells and a pharmaceutically acceptable 14 carrier such as physiological saline, non-toxic buffers and the like.

16 Means for detecting tumor aggressiveness and/or 17 metastatic activity is now also made possible by a kit 18 comprising separate containers containing antibodies 19 having specific binding affinity Zor AMF; labelled AMF; unlabelled AMF and instructional material for 21 performing tests utilizing the antibodies and the AMF 22 provided in the kit for determining AMF and/or receptor 23 activity in a body sample. Such accessories as 24 microtiter plates, micropipettes, moans for reading antibody titer ar..l the like are routinely found in such 26 kits and may be included for convenience in tho kits of 27 the present invention.

28 In summary, the present invention provides a now 29 tool for understanding mechanisms which control tumor cell invasion and opens new strategies for cancer 31 diagnosis and therapy, Epitholial cells do not normally 32 exhibit invasive behaviOr, The motility factor described 33 herein does not affect the migration of normal blood 34 loukocytes. Therefore, a therapeutic agent aimed at 7) WO 88/09797 PCT/US88/01805 1 inhibiting the factor described in the invention should 2 have low toxicity against normal resting tissues.

3 Pharmacologic preparations obtained in accordance with 4 the present invention which inhibit invasion of tumor cells and prevent the transition from in situ to invasive 6 carcinoma could be potent cancer arresting agents.

7 Inhibitors of tumor invasion can also prevent the growth 8 of established metastases because a metastasis may need 9 to invade locally as it grows. Furthermore, such agents may inhibit tumor angiogenesis. Antibodies to motility 11 factors or their receptors could be applied through 12 tissue immunohistology, radioscintography, or serum 13 immunoassays to localize metastases and predict cancer 14 aggresseiveness in individual patients. As gene products, autocrine motility factors or their receptors 16 define a new class of oncogenes. The level of expression 17 of these genes in a patient's tumor may provide tmportant 18 diagnostic information through monitoring the level of 19 AMF in the body sample.

Of course, invasion and metastases are among the 21 major causes of cancer treatment failure. The present 22 invention provides new clinical strategies to detect 23 pro-invasive lesions and prevent their progression; (b) 24 accurately predict the aggressiveness of a patient's tumor, and identify and eradicate micromotactases.

26 One of the least understood aspects of tumor invanion is 27 tumor cell locomotion. The present invention allows the 28 determination of the role of the tumor cell motility 29 factor.

It is understood that the examples and embodiments 31 described heroin are for illustrative purposes only and 32 that various modifications or changes in light thereof 33 will be suggested to persons skilled it. the art and are 34 to be included within the spirit and purview of this application and scope of the appended claimsb

Claims (5)

1. Antibodies having specific affinity for autocrine motility factor (AMF) said AMF having at the NH 2 terminus, an amino acid sequence at least in part as follows: DKELRFRDCTKSLAEANKK
2. A kit for detecting tumorogenic or metastatic activity in a body, wherein said kit includes a container containing antibodies having specific binding affinity for autocrine motility factor (AMF).
3. A kit for determining the level of AMF cell receptors wherein said kit includes containers separately containing labelled AMF; unlabelled AMF; and instructions set* for performing tests with a body sample to determine the level of AMF-receptor activity. Anti-AMF antibodies which inhibit tumor cell motility. A method of detecting the presence of cancer in SOOhumans comprising the step of reacting a human body sample suspected of having cancer with the antibody of claim 4, a positive result being indicative of the presence of cancer in said patient.
6. Anti-AMF antibodies as hereinbefore described with reference to the section of specification titled "Materials Methods".
7. A method of detecting the presence of cancer in S• ?r humans as hereinbefore described with reference to the section of the specification titled "Materials Methods". DATED this 12th day of June 1991 e e THE UNITED STATES OF AMERICA, as represented by the SECRETARY, U.S. DEPARTMENT OF COMMERCE Patent Attorneys for the Applicant: F.B. RiCa CO. 400
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