CA1310902C - Autocrine motility factors in cancer diagnosis and management - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention described an isolated and substantially pure mammalian cell polypeptide which stimulates random locomotion of producer cell and which has a molecular weight greater than 30,000. The unique polypeptide of the present invention is inhibited by pertussis toxin. A kit and method for detecting metastastis in human are also described.

Description

13~0902 2 DIAGNOSIS AND MANAGE~ENT

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 complex cascade of invasion 13 and metastases. Such stages include the detachment and 14 subsequent inflitlation 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 ln 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 131~902 ] chemoattractants. Todaro, et al. (Proc. Natl. Acad. Sci 2 USA, 77:5258-5262, 1980) reported autocrine growth 3 factors for transformed cells Other gro~th factors of 4 various types are also kno~m. 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.

12 It is, therefore, an object of the present invention 13 to identify and provide an autocrine factor controlling 14 motility of tumor cells, such autocrine 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, localizing and 21 predicting metastases and tumor angiogenesis in humans.
22 It is yet another ob;ect 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.

131~902 1 Various other ob~ects and advantages of the present 2 invention will become evident from the Deta~led 3 Descr~ption of the Invention.

, .
4 BRIEF DESCRIPTION OF TH~ DRAWINGS

These and other objects, features and many of the 6 attendant advanta~es 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 (a) Scatchard analysis of lZ5I-AMF
13 binding to suspended tumor cells; and (b) dose response 14 curve of cell motility to purified AMF.

DETAILED DESCRIPTION OF THE INVENTION

16 The above and various other ob~ects and advantages 17 of the present invention are achieved by a polypeptide 18 having the following properties: (a) secreted by 19 mammalian cells and stimulates random locomotion of the producer cells: (b~ having molecular weight of > 30,000;
21 and (c) 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 lts NH2 terminus (single letter code) or at the NH2 terminus of an active fragment of the 26 polypeptide:

28 This invention is specifically concerned with .~ '.

131~19~2 -3a-1 antibodies having specific binding affin~ty for autocrine 2 motility factor (AMF) having at the NH2 terminus, an amino 3 acid sequence at least in part as follow~:
4 D X E L R F R D C T K S L A E A N K K.

131L(39~32 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 m~terials 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.

MATERIALS AND METHODS

11 Cell Lines 12 Human MD~231 and MDA435 breast carcinoma cells lines 13 were obtained from ATCC and cultured in Dulbecco's 14 modified Eagle's medium ~DMEM) supplemented with 10~
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 18 5 x 105 cells into the lateral tail vein.

lY 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 H2O. This solution is applied to a PD-lO ~Sephade~ G25 medium) column. The 26 first 2.5 ml are discarded and the next 4 ml are 27 collected. The effluent contains AME 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 in 31 PBS (source of column). Elution with PBS
*Trade Mark 13~9~2 1 yields an active fraction that corresponds to material 2 with a molecular weightof about 54 kDa. This fraction is 3 dialyzed and concentrated 25 fold. The material is made 4 up to 50 mM Tris-acetate, pH 8.0 and applied to a mono Q
anion exchange column (source) and eluted with a linear 6 salt gradient (0-1 M NaCl) with the following 7 modification: When the NaCl concentration reaches 8 0.25 M, this concentration is held for 10 min before 9 resuming the gradient. AMF is eluted in the 0.3 M to 0.4 M NaCl fraction. The active fraction is dialyzed 11 and concentrated to a small volume (about 0.5 ml) which 12 in turn is made up to 0.02 M phosphate in normal saline, 13 pH 7.4. This is applied to a heparin column in PBS. The 14 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 16 gradient. After each purification step, column fractions 17 (dialyzed to remove salt) are assayed for motility 18 stimulating activity by the modified Boyden chamber 19 procedure.

Assay Procedure for Cell Motility 21 The assay of motility is accomplished b~ the use of 22 a modified Boyden (Zigmond, et al, J. Exp. Med.
23 137:387-410, 1973) chamber. This is a device (Figure 1) 24 consisting of 2 wells horizontally separated by a microporous polycarbonate filter with a pore diameter of 26 about 8 u. The motility stimulus (or chemoattractant) is 27 placed in the lower well to contact the filter. To the 28 upper well is added a suspension of cells (for example 29 A2085 melanoma cells) at a concentration of about 106 cells/ml. The chamber is then placed in a humidified 31 incubator for about 4 hours at 37 degrees C in an 32 atmosphere of air and about 5~ C02. During this time, 33 the cells are deposited by gravity on the topside 13~0902 1 of the filter. However, some cells (about 5 to 10~) 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 period, the filter is removed and 7 subjected to a fixing 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. T:le 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 ocular, the cells in 5 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) Motility 26 Measurement of random motility is accomplished by 27 exposing the cells to a fixed concentration of stimulus 28 and determining their migration as described above. This 29 includes adding e~ual increasing concentrations of attractant to both upper and lower wells prior to the 31 assay incubation. The random migration of cells as a 32 function of the levels of attractant is then determined.
33 Directed migration occurs if the cells migrate better in 131~02 1 positive gradients (higher concentrations of attractant 2 in the lower well compared to the upper well) than in 3 negative gradients (higher concentrations in upper well 4 than in lower well). The results of such an assay are shown in the "checkerboard" tabulation of the results 6 (Table 1). It can be seen that random motility is quite 7 significant for the A2~58 melanoma cells responding to 8 the A~F.

% Motility Eactor in upper Well % Motility Factor In 15 494 1056 825 1469 452aoo 2550 2262 'diagonal' shows random migration of cells. Lower triangle shows directed migration of cells in a positi~e gradient of motility stimulus.

- 13109~2 -- 8 ~

1 Assay for Cell Pathways Invol~ed in AMF Induced Motility 2 MateriaLs: DMEM supplements with L-glutamine (2 ~g), 3 penicillin and streptomycin with or without 10~
4 heat-inactivated fetal calf serum were purchased from commercial sources s~ch as Meloy Laboratories, Inc.
6 (Springfield, VA). Pertussis toxin and cholera toxin 7 were obtainea from List Biological Laboratories, Inc.
8 (Cambell, CA). Phorbol 12-myr~state 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) 2S 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), A20S8 cells were innoculated for 48 hours in DMEM
24 without any protein supplement. The medium was concentrated us~ng 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 30 accordance with this techni~ue 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 *Trade Mark B
.

~310902 g 1 2 x 106/ml in DM~M with 1 mg/ml bovine serum albumin.
2 The assay was performed in 48-well micro-chemotaxis 3 chamber (Harvath et al, 1980 supra) with 8 ~m Nucleopore 4 membranes coated with type IV collagen. The chambers were incubated at 37 degrees C for 4-5 hours, then 6 devel~ped using Diff Quick stains (American Scientific).
7 The stained membranes were placed onto glass slides with 8 the original cell side up so t~at the cell pellet could 9 be wiped ~rom 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 13 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 chemokinetic 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 ~) 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 PsS 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 sera control. Purified AMF protein (10 31 ~g) was emulsified in complete Freundi's adjuvant and 32 in;ected into a subcutaneous site on the back of New 33 Zealand white rabbits. Booster injections of 5 ~g were 131~902 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 56C for 30 minutes.

6 Determination of AMF Purity 7 The purity of the isolated AMF was determined by the 8 following criteria:
9 (a) 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 (b) Protein band cut from the gel retains motility 14 stimulating activity.
(c) NH2 terminus amino acid sequence (1-19) reveals one 16 type of amino acid residue at each cycle; and 17 (d) 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 AMF is performed with the Applied Biosystems (Foster City, CA) model 470A
26 gas-phase sequencer using the trifluoracetic acid 27 chemistry provided by the manufacturer. The 28 phenylthiohydantoin amino acids were identified and 29 quantitated by using the Perkin~-Elmer series 3B HPLC and ultraviolet detection.

13~ 09~2 1 Dose Response 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 ~g/ml. Cell viability 7 at any of the tested doses was comparable to the 8 viability in untreated control (>90%). 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 2). Preincubation for 30 minutes to 16 2 hours at doses of 0.5 - 1.5 ~g/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 descrlbed inhibitory doses of PT for Gi and Go 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.

TABL~ 2 AMF TREATMENT DATA

_ Treatment Motility (~ of Controls) 1 P'ase K 13.2 2 DNAase 2 g/ml95.1 3 RNAase 104 4 PMSF 5 mM 95.5 DDT 10 mM 11.5 6 Heating 100C 5.0 7 Heating 56C 97.2 8 pH 4.0 20 9 pH 7.4 100 pH 11.0 100 P~RTUSSIS TOXIN INHIBITION OF AMF INDUCED MOTILITY
B. Time Pertussis Toxin Added Percent Inhibition (hrs. from start of of AMF Induced assay) Motility -2.0 100 -1.0 95 -0.5 100 Start of Assay* 0 62 +0.5 55 +1.0 33 +2.00 (no inhibition)*+
+3.00 (no inhibition)*+

*Time of addition of AMF
*+Pertussin Toxin requires at least 1 hour to penetrate cell membranes and inhibit G proteins by ADP ribosylation.

~31~902 1 _olera Toxin Dose Response and Time Course: Cholera 2 toxin (CT) in contrast to pertussis toxin, is thought to 3 act on the Gs protein that stimualtes adenylate cyclase 4 to produce the second messenger, cAMP. Cholera toxin was added to A2058 cells either for overnight incubation in 6 flasks or for variable periods of preincubation prior to 7 the start of the chemotaxis assay. The tested doses of 8 cholera toxin ranged from about 0.1-50 ~g/ml. At all 9 tested doses, cell viabllity was comparable to that of untreated cells (>90%). Treated and untreated cells were 11 then tested for chemotactic response to A2058 conditioned 12 medium.
13 Overnight treatment with CT caused a diminished 14 response to the A2058 conditioned medium,though the inhibitory effect was never complete (30-60~
16 inhibition). If the cells were exposed to cholera toxin 17 for just a brief preincubation prior to the start of the 18 chemotaxis assay, the inhibition was minimal (<5~).

19 Effect of Other Agents Involved in the Adenylate cyclase System on Cell Motilit~: Cholera toxin is thought to act 21 by ADP-ribosylatlon of the Gs protein in an active 22 configuration that can stimulate adenylate cyclase.
23 Since the effect of cholera toxin on A2058 cell motility 24 was minimal, further tests were conducted to determine whether other agents that act on the cAMP pathyway would 26 be inhibitory. Forskolin stimulates adenylate cyclase 27 directly without acting through an intermediary G
28 protein. The cAMP analogue, 8-Br cAMP, is able to enter 29 intact cells. Both chemicals were added to A2058 cells either for overnight incubation in flasks or for a 2 hour 31 preincubation prior to the start of chemotaxis. Both 32 exhibited only a partial inhibition of cell motility that 131~9~2 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 concentratio-~ of con~itioned 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 well 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 cellular or serum 21 components could activate or inhibit the action of the 22 motility factor. The motility factor is inactivated by 23 exposure LO ~treptococcal protease, but active 24 chymotrypsin-derived fra~ments can be produced (data not shown). The activity is 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 chemoattractants.
31 It was also found that known growth factors such as 32 PDGF, ~TGF, ~TGF, EGF, IGF, transferrin, or FGF do not 33 substitute or block the AMF (data not shown). Amino acid 34 analysis indicated a unique sequence of 19 amino terminal 1 amino acids of AMF. A slightly small form of the 2 active material was also found to have a unique amino 3 terminal ~equence. Protein data base searches failed to 4 reveal any ~ther polypeptide with such a se~uence.
It has also been found that motility induction by 6 AMF is not blocked or substituted by known growth factors 7 or serum factors. At a concentration of l nM or less, B AMF markedly stimulates the random and directed motility 9 of breast cancer cells but fails to induce motility in leukocytes. The factor also stimulated random pseudopodia ll production by breast carcinoma cells and melanoma cells.
12 Following trasfection with the activated ras-oncogene, 13 AMF and its receptor are enhanced more than lO0 fold in 14 certain cells. Human breast carcinoma cells, but not normal breast epithelium, produce large quantities of 16 AMF. Antibodies recognizing AMF abolish human tumor cell 17 motility in vitro without altering tumor cell viability.
18 The availability of an isolated and purified l9 autocrine, polypeptide, tumor motility factor makes it possible to obtain anti-AMF antibodies having specific 21 binding affinity for said motility factor. Such 22 antibodies can either be polyclonal or monoclonal and are 23 prepared by well known standard techniques routine in the 24 art. Such antibodies can also be labelled with suitable radioisotopes or fluorescent and other markers or ligands 26 and employed for the detection, quantitation and/or 27 localization of the AMF in human tissue or body fluid.
28 Furthermore, radiolabelled AMF together with unlabelled 29 AMF can be utilized in a standard competitive assay to measure AMF receptor level. Such binding assay for 31 determining the receptor level is carried out as follows.

AMF B i~5~s say:
2 Purified AMF lS lodinated using the standard ~io Rad 3 e~zymobead proce~ure. Increasing amounts of labeled AMF
4 is incubated in a volume of 1 ml with 100,000 A2058 melanoma cells, in the presence or absence of 100 fold 6 excess cold competitor. Incubation is conducted at 37C
7 for 40 minutes and the cell-bound radioactivity is 8 separated by centrifugation. AMF binding exhibits 9 saturation with 80~ specific binding and about 30,000 receptors per cell. Scatchard analysis according to 11 standard methods shows a linear relationship between the 12 specifically bound/free ratio and the specifically bound 13 AMF, with an estimated kd in the range of about 0.5 nM.
14 Detection of cancer in humans is also made possible by the present discovery and testing of human body 16 samples for this purpose is now illustrated using urine 17 samples from bladder cancer patients.
1~ Urine samples from patients with bladder cancer are 19 collected and processed with centrifugal microconcentrator (AMIC0~) with an excluslon filter of 10 21 kilodaltons. The processed urines are reconstituted at a 22 10-fold concentration with steriel phosphate buffered 23 saline pH7.5 and stored at -20C until use. Tumor grade`
24 is determined by a pathologist using a scale of one to three with grade one tumors showing the most 26 differentiation and grade three tumors showing the least 27 differentiation. Bladder tumors are staged according to 28 the American Joint Committee TNM classification.

29 Assay of Urine Samples:
Although any cell line which responds to AMF can be 31 employed, the preferred cell line is human MDA 435 cells 32 (ATCC). The concentrated ur~ne samples are applied to the 33 microwell migration chamber assay as described herein *Trade Mar]c '.

~3109~2 1 supra. Each sample is tested at a series of dilutions 2 with and without the addition of the antibodies directed 3 against human tumor AMF. AMF units are recorded as the 4 proportion of tumor cells stimulated to migrate by the sample which is inhibited by the antibodies. In general, 6 greater than 80% of the stimulated migration is inhibited 7 by an antibody concentration of about 10 ~g/ml.
8 As shown in Table 3, control urines with 9 non-neoplastic disorders such as kidney stones failed to contain significant levels of motility factors. All of 11 the bladder transitional cell carcinoma cases exhibited a 12 positive motility response in the urine. The highest 13 levels of motility factor production was found in the 14 urine of patients with high grade tumors or with stage D
(metastatic) tumors.

. .

i31~902 Urine SampleAMF units SE
1 Control ks 75 5 5 2 Control ks 76 9 2 3 Ca in Situ 32 5 4 Papillary TCC64 3 8 Recur TCC 79130 22 13 TCC stg D 584234 25 TCC = Transitional cell carcinoma of the bladder Recur TCC = Recurrent TCC
TCC II = grade II
TCC III = grade III
TCC stg D = metastatic TCC
KS = Kidney stones SE = Standard error 131~9~2 l Of course, the antibodies against AMF can be 2 employed to block or inhibit AMF activity thereby 3 arresting tumor invasion or metastatic proliferation 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 8 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 (a) antibodies l9 having specific binding affinity for AMF; (b) labelled AMF; (c) 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 samp~e. Such accessories as 24 microtiter plates, micropipettes, means for reading antibody titer and the like are routinely found in such 26 kits and may be included for convenience in the kits of 27 the present invention.
28 In summary, the present invention provides a new 29 tool for understanding mechanisms which control tumor cell invasion and opens new strategies for cancer 31 diagnosis and therapy. Epithelial cells do not normally 32 exhibit invasive behavior. The motility factor described 33 herein does not affect the migration of normal blood 34 leukocytes. Therefore, a therapeutic agent aimed at 131~902 1 inhibiting the factor described in the invention should ~ have low toxicity against normal resting ~issues.
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 o~ established metastases because a metastasis may need 9 to invade locally as it grows. Furthermore, such agents may inhibit tumor angiogenesis. Antibodies to motility ll 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 important 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 (a) detect 23 pre-invasive lesions and prevent their progression; (b) 24 accurately predict the aggressiveness of a patient's tumor, and (c) identify and eradicate micrometastases.
26 One of the least understood aspe-ts of tumor invasion 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 herein iare for illustrative purposes only and 32 that various modifications or changes in light thereof 33 will be suggested to persons skilled in the art and are 34 to be included within the spirit and purview of this application and scope of the appended claims.

Claims (3)

1. Antibodies having specific binding affinity for autocrine motility factor (AMF) having at the NH2 terminus, an amino acid sequence at least in part as follows:

D K E L R F R D C T K S L A E A N K K.

2. A method of detecting the presence of cancer in humans comprising the step of reacting a human body sample suspected of having cancer with cells which become motile under the influence of autocrine motility factor (AMF) with the antibody of claim 1, a positive immunological result being indicative of the presence of this type of cancer in said patient.

3. A kit for detecting tumorgenic or metastatic activity with cells which become motile under the influence of autocrine motility factor (AMF) in a body, comprising a container containing antibodies having specific binding affinity for autocrine motility factor (AMF) having at the NH2 terminus, an amino acid sequence at least in part as follows:

D K E L R F R D C T K S L A E N K K