JPH04502143A - Autocrine motility factors in cancer diagnosis and management - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Autocrine motility factor technology in cancer diagnosis and management FIELD OF THE INVENTION The present invention relates generally to the field of cancer diagnosis and management.

More specifically, the present invention provides novel tumor motility factors and cancer diagnosis, prevention and treatment. on their use in devising new approaches to.

Background technology Cell motility is involved in many steps in the complex cascade of tumor cell invasion and metastasis. is necessary to cross the

Such steps include - subsequent detachment of cells from the tumor into adjacent tissues and subsequent infiltration; Migration of cells through blood vessel walls into the circulation (intravascular foreign body invasion) and secondary sites of cells Extravasation to the blood vessels has been mentioned. Biological barriers such as the endothelial basement membrane of the vasculature Movement of cells through cells can occur by chemotactic mechanisms. some tumors Studies on in vitro chemotaxis of cells have been conducted using various compounds, such as complement inducers, Connective tissue components consisting of largen peptides, formyl peptides, and chemoattractants This shows that it can act as a Todaro et al. (Proc, Nat 1゜Aca d, Sc i USA, 77: 5258-5262゜1980) was converted reported an autocrine growth factor for cell use. Various other growth factors are also known. Ru. However, tumor cell chemotactic motility (tropism) and chemokinesis ( The existence and role of autocrine factors that control random) motility have not been previously known or is not listed. Cell movement is different from cell growth and proliferation in terms of cell behavior. It may be important to note one thing here.

Disclosure of invention Therefore, it is an object of the present invention to identify autocrine factors that control tumor cell motility (such as The purpose of the present invention is to identify and provide an autocrine factor, herein referred to as "AMFJ."

Yet another object of the present invention is to The objective is to provide antibodies that

Yet still another object of the invention is to detect metastasis and tumor angiogenesis in humans; The object of the present invention is to provide a kit for localizing and predicting.

Yet another object of the invention is to predict and prevent metastatic invasion and cancer growth in humans. and/or to provide a method for treatment.

An additional object of the present invention is to inhibit tumor cell motility in a pharmaceutically acceptable carrier. An object of the present invention is to provide a pharmaceutical composition containing an effective amount of neutralizing antibodies against AMF. .

Various other objects and advantages of the invention will become apparent from the detailed description of the invention. Probably.

Brief description of the drawing Many of these and other objects, features and attendant advantages of the present invention will be apparent from the accompanying drawings. will be better understood when reading the specific explanation below when considered together with the Figure 1 shows a schematic diagram of the Boyden test; Figure 2 shows (a) suspended tumor cells; Scatchard analysis of 125I-AMF binding to; and (b) purified AMF Figure 3 shows dose-response curves for cell movement.

BEST MODE FOR CARRYING OUT THE INVENTION The foregoing objects and various other objects and advantages of the present invention include Achieved by putide = (a) Secreted by Eltli mammalian cells and stimulates random migration of productive cells; (b) has a molecular weight >30,000; and (C) inhibited by pertussis toxin. The polypeptide of the present invention comprises at least may also be partially or completely linked to the NH2 terminus (single letter code) or the activity of the polypeptide. The NH2 terminus of the sex fragment was found to have the following amino acid sequence order: Ta: DKELRFRDCTKSLAEANKK Used here unless otherwise specified. All technical and scientific terms have the same meaning as commonly understood by those skilled in the art. Has taste. Any methods and materials similar or equivalent to those described herein may be used. Although methods and materials can also be used in the practice or testing of the present invention, preferred methods and materials are preferred. I will now explain the fees. All publications mentioned below are incorporated herein by reference. .

Human MDA231 and MDA435 breast cancer cell lines were obtained from ATCC and supplemented with 10% bovine Cultures were in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal serum.

Both of these estrogen-independent cell lines were injected into the lateral tail vein with 5 x 105 cells. Metastases develop in the lungs of 6-week-old NIH nude mice 6 weeks after injection of the cells.

Isolation and purification of autocrine motility factors MDA231 and MDA435 human breast cancer cells were grown in DM in the absence of added proteins. Grows to 60% confluency in EM. Lyophilize the medium and distill the residue Dissolve in approximately 2 ml of H2O. This solution was added to PD-10 (Sephadex G25 medium). ) column. Discard the first 2.5 ml and collect the next 4 ml. Effluent contains AMF separated from low molecular weight substances. This collected fraction was 0.02M phosphate buffered saline (PBS) at pH 7.4 Water and apply to a Sephacryl S-300 column in PBS (column source). P Elution with BS yields an active fraction corresponding to a substance with a molecular weight of approximately 54 KDa. . This fraction is dialyzed and concentrated 25 times. This substance was added to 50mM trichloride at pH 8.0. acetate, applied to a MonoQ anion exchange column (source), and made the following modifications. Elute with a linear salt gradient (0 to IM NaCl): NaCl concentration is 0.25M When reached, this concentration is held for 10 minutes before taking the gradient. AMF to 0 ．． Elutes in the 3M-0.4M NaCl fraction. The active fraction was dialyzed and a small volume ( Concentrate to approximately 0.5 ml). 111 condensate in normal saline at pH 7.4. M phosphate. Apply this to a heparin column in PBS. The column is , a linear gradient of NaCl (0 , 15M to IM).

After each purification step, the column fraction (dialyzed to remove salts) was modified with Boydenchi. Assay for locomotor stimulation activity by the Ember method.

Cell movement assay method The motility assay was performed using a modified Boyden method (Zsigmond et al., Exp. Med, 137). : 387-410. 1973. ) achieved by the use of a chess bar. child This is carried out horizontally by a microporous polycarbonate filter with a pore size of approximately 8μ. This device (Fig. 1) consists of two wells separated into two wells. Motility stimulants (also (chemoattractant) is placed in the lower well and brought into contact with the filter. in the upper well Suspension of cells (e.g. A2085 melanoma cells) at a concentration of approximately 106 cells/ml Add liquid. The chamber was then heated at 37°C in an atmosphere of air and approximately 5% CO2. Place in a humidified incubator for approximately 4 hours. At this time, cells are filtered by gravity. - is deposited on the upper side of the However, some cells (approximately 5-10%) are motile Migrate to the lower side of the filter in response to sexual stimulants. The average diameter of the cells is smaller than the pore size. Since it is large, energy consumption must occur during movement. culture period At the end, the filter is removed and subjected to a fixation staining procedure. First of all, this Soak the router in the methanol-containing solution for about 2 minutes, then in the eosin solution for 2 minutes. immersion and then immersion in a hematoxylin solution for 3 minutes. after that , wash the filter in water and place it top side up on a glass slide. upper side Completely remove the button of stained cells on top with a small piece of dry tissue paper. Next The stained cells that have migrated through the filter are revealed. Approximately 500 of these Count by microscopy at a magnification of X. 5 different high power fields in one Visualize with a grid in the eyepiece and count cells in five fields. Count and calculate the average to 21. Positive regulation/negative regulation ratio ≧5 indicates cell motility stimulation Indicates a positive response to the drug.

Measurement of Random and Directed (Chemotactic) Motility Measurement of random motility is a Achieved by exposing to a fixed concentration of stimulant and measuring migration as described above . This means that equal increasing concentrations of attractant are applied to the top and bottom wells before assay incubation. This includes adding to both the part well and the part well. Then, as a function of the amount of attractant Measure the random movement of cells. Directed migration allows cells to move up a negative gradient (from the bottom well positive slope (higher concentration in the upper well) than in the lower well compared to the bipartite well. This will occur if C (attractant) moves better at higher concentrations in the wells. It takes The test results are shown in the “Cheetsker board” table of results (Table 1). 6 Random interlocking is AM It can be seen that it is quite significant in the case of A2058 melanoma cells responding to F.

Table 1 Motility factor % in upper well Bottom U　0! 00 244 512 494 15 494 1058 825 1469 30 1781 1550 2144 2640 luck Mobility 45 2800 2550 2262 43G2 Prisoner 06 "Diagonal" indicates random movement of cells. The lower triangle indicates the positive side of the motility stimulant. shows directed migration of cells in a gradient of .

Assay material for cellular pathways involved in AMF-induced motility: L1-glutamine (2 μg ), 10% heat-inactivated baby serum with penicillin and streptomycin DMEM supplements with or without commercially available sources, such as Meloy Laboratories Purchased from Springfield, VA. Ta.

List of 100 [1g toxins and cholera toxins] Biological Laboratories I Retrieved from Incorporated (Bell, Gear, Calif.). Holbor 12-myristate 13-acetate (PMA), phorbol 12°13-di Decanoate (PDD), calcium ionophore A23187, diltiase Nifedipine, Verapamil, Trifluoberazine, Leupeptin, Fors All choline and 8-Br cAMP were purchased from Sigma Chemical Company (Mi Purchased from St. Louis, Zuri). 1-oleoyl-2-acetylglycero was purchased from Molecular Probes (Eugen, Oregon). Nucle Opoa@(polyvinylpyrrolidone-free) and 48-well chemotaxis chain Neuroprobe Inc. (Cabin, Maryland) Purchased from John.

Cell culture: Human melanoma cell line A2058 was cultured as described by Todaro et al. Maintained.

(Liotta et al., Proc, Nat 1. Acad.

A2058 cells were seeded in DMEM for 48 hours without protein supplementation. molecular weight media using a cut-off 30,000 Dalton Centricon ultrafiltration assembly. was concentrated.

Chemotaxis assay: The assay used to measure cell movement is described by Barbas et al. (1980 (1986), a modification of the technique described by Barbas et al. (1986), supra.

According to this technique A205g, melanoma cells (approximately 75-90% of the entire surface) are trypsinized. - Harvested with EDTA and placed in DMEM supplemented with 10% fetal bovine serum at room temperature. It was collected for at least one hour. The cells were then incubated with bovine serum albumin 1 μ/ml. Resuspend in DMEM at 2X106/ml. The test is coated with type ■ collagen. 48-well microchemotaxis chamber with 8 μm nucleopore membrane (previously (Barbas et al., 1980). Incubate the chamber at 37°C for 4-5 hours. and Diff-Quick stain (American Scientist). The color was developed using As the cell pellet is blown off the surface The stained membrane was placed on a glass slide with the original cell side up. through the pores The migrated cells are trapped between the glass and the membrane, and a high power field (50 0X) and could be easily counted by optical microscopy. Unstimulated random movement is different from directed movement. <20% of the movement.

Before or during chemotaxis assays, chemicals can be co-incubated with cells to alter cell metabolism. or stimulate a chemokinesis response. At the beginning of the test, chemicals We were also able to demonstrate chemotactic potential in addition to the lower chamber.

Production of murine antibodies against AMF Purified AMF protein (10 μg) was emulsified with Freund's complete adjuvant, C3H mice were injected intravenously. Two weeks later, mice were injected into the tail vein in a volume of 0.1 ml. The mice were supplemented with 5 μg of AMF in PBS injected intravenously.

One month later, the mice were exsanguinated and the serum was tested for its ability to inhibit tumor cell movement. Ta. In this assay, mouse serum was tested for A Precultured with MF. At a dilution of 1/1000, mouse sera were pooled. This resulted in a 90% inhibition rate of tumor cell movement compared to the mouse serum control. spirit AMF protein (10 μg) manufactured by AMF was emulsified in complete Freund's adjuvant and added to garlic. - Injected subcutaneously on the back of a Zealand White Rabbit. booster injection 5 μg of solution was applied at 6 and 12 weeks. At the 3rd and 4th month, the rabbits are released. Blood and serum were tested for motility-inhibitory activity.

At a dose of 1/1000, the immune serum was compared to the control pre-immune serum. This destroyed their motility. Serum was heat inactivated at 56°C for 30 minutes.

Determination of AMF purity The purity of isolated AMF was determined by the following criteria: (a) A single 54 kDA band was detected in the first order performed by standard methods well known in the art. Found in original and two-dimensional polyacrylamide gel electrophoresis. Protein is Identified with silver stain.

(b) Protein bands cleaved from the gel retain motility-stimulating activity.

(c) N H2 terminal amino acid sequence order (1 to 19) in each cycle One type of amino acid residue is shown.

(d) Murine and rabbit anti-AMF antibodies stimulate the motility-stimulating activity of human tumor AMF. Cut off.

Based on the above criteria, the isolated AMF of the present invention was found to be substantially pure. It was done. As used herein, the term "substantially" refers to It means being as pure as possible.

Amino acid sequence ordering is Erasing purified AMF using the trifluoroacetic acid chemistry provided by the manufacturer. Applied Biosystems (Foster Systems, Calif.) T) Performed on a Model 470A gas phase sequencer. phenylthiohydant In-amino acids were analyzed using Parking-Elmer Series 3B HPLC and ultraviolet rays. were identified and quantified by using

Pertussis toxin dose response and time course and effects on motility: pertussis toxin ( PT) for overnight culture in flasks for various periods of pre-incubation before assay. A2058 was added to A2058 for the first time or at different time points after the start of the assay.

The PT doses tested were approximately 10 ng/ml to 1.5 ug/ml. tested The cell viability at any of the doses tested was the same as the viability in the untreated control ( >90%). The treated and untreated cells were then conditioned to A2058. Tested for motility response to medium. Cell movement in response to DMEM alone , were included as negative controls for each treatment group of cells.

Overnight culture of cells with either of the tested FT doses resulted in significant inhibition of cell motility. (Table 2). Preincubation for 30 minutes to 2 hours at a dose of 0.5 to 1.5 μg/ml also Resulted in inhibition higher than 50%. Add pertussis toxin at the beginning of the assay or after the assay begins. There was a gradual decline in the inhibitory effect when 1 to 2 hours after starting the assay, P T had minimal effect on observed motility.

The dose response of PT is consistent with the amount of inhibition of PT for G and Go proteins. did. The time course may be due to adding inappropriate doses of PT or saturating G protein sites. showed much reduced inhibition when insufficient time was applied. Therefore, this test The experimental data show that AMF requires 6 proteins to activate cells. This was consistent with the hypothesis that cell movement is stimulated through receptors that stimulate cell movement.

I　P’aseK　13.2 2 DNAase 2g/ml 95.13 RNAase 104 4 PMSF 5mM 95.5 5 DD710mM 11.5 6 Heating 100℃ 5. 0 7 Heating 56℃ 97.2 8 pH 4,020 9pH7,4100 10pH11,0101 Pertussis toxin suppression of 00A-induced motility Addition of pertussis toxin Time (test start? AMF-induced motility Suppression rate of B et al.'s hr) -2,0100 -1,095 -0,5100 Test start* 0 62 +2.0 0 (no suppression) ★+ +3.0 0 (no suppression) ★+ ★AMF addition time ★+ Pertussis toxin requires at least 1 hour to penetrate cell membranes and ADP Inhibits G proteins by ribosylation.

Cholera toxin dose response and time course of cholera toxin (CT) in contrast to bipertussis toxin adenylate cycler to produce the second metsenger, cAMP. It is thought that it acts on the G protein that stimulates enzymes. cholera toxin, flask for overnight incubation or for preincubation for a variable period of time before starting the chemotaxis assay. was added to A2058 cells. The test dose of cholera toxin is approximately 0.1 to 50 μg. /ml. At all doses tested, cell viability was Cell viability (>90%) was comparable. The treated and untreated cells were then placed in A2 The chemotactic response to 058 conditioned medium was tested.

Although the suppression effect is never complete (inhibition rate 30-60%), overnight treatment with CT reduced the response to A2058 conditioned medium 1:. Start cell chemotaxis assay If exposed to Corinola toxin for a short period of time immediately before incubation, inhibition is minimal. (5%).

Effects of other drugs involved in the adenylate cyclase system on cell motility: cholera The a-element is a G protein in an active configuration that can stimulate adenylate cyclase. It is believed that it acts through ADP ribosylation.

Because the effect of cholera toxin on A2058 cell motility is minimal, further tests experiments to determine whether other drugs acting on the cAMP pathway are inhibitory. did.

Forskolin inhibits adenylate cyclase without acting through mediating G proteins. stimulate directly. c　A　MP　analog, 8-Br　cAMP can enter the cell. Both chemicals for overnight culture in flasks. , or added to A2058 cells for a 2-hour preincubation before the start of chemotaxis. both Both showed a partial inhibition of cell motility that was essentially identical to the inhibition of cholera toxin for a comparable period of time. Only the system was shown.

These cells are obtained by culturing confluent cells in serum-free medium. Motility factors respond in a dose-dependent manner to various concentrations of conditioned medium. was concluded to be derived from cells. Obtained in a modified Boyden chamber experiment The results show that the autocrine factor of the present invention has chemotactic (directional) and chemokinetic (random) properties. We also demonstrate that it has both motility and motility. Random stimulation is more important than directed movement. Since the cells were found to be approximately 3 times larger, they were exposed to gradients of motility factors as well as attractants. It was concluded that it responds to high homogeneous concentrations of .

When measured by gel filtration and gel electrophoresis, the migration stimulators of the invention are , was found to have a molecular weight of approximately 54 kilodaltons. This form is the active factor It may be a precursor of. Cellular or serum components activate the action of motility factors. It is possible to sexualize or suppress.

Motility factors are inactivated by exposure to streptococcal protease, but remain active. chymotrypsin-derived fragments can be produced (data not shown). The activity is It is destroyed by exposure to temperatures of 56°C, but is stable upon exposure to 56°C.

Additionally, the activity is stable in the pH range 4-11 (data not shown). these The nature of the autocrine substance (AMF) of the present invention is that it is compatible with various known growth factors and chemical inducers. Indicates that it is different from the attracting substance. In addition, known growth factors such as PDGF, α TGF, βTGF, EGF, IGF, transferrin or FGF is AMF (data not shown). Amino acid analysis The unique sequence order of the 19 amino-terminal amino acids of AMF is shown. slightly smaller It has been found that different forms of the active substance also have unique amino-terminal sequence orders. Ta Protein database searches indicate other polypeptides with such sequence order. I lost it.

In addition, motility induction by AMF can be blocked by known growth factors or serum factors. or found not to be replaced. At concentrations below 1 nM, AMF is , significantly stimulates random and directed motility of breast cancer cells, but has no effect on leukocytes. failure to induce motility. In addition, the factor stimulated pseudopod production. After transfection with active ras oncogene, AMF and its receptors are increased more than 100-fold in cells consisting of human breast cancer cells (but not normal breast epithelium) produce large amounts of AMF.

Antibodies that recognize AMF can improve human tumor cell motility in vivo without altering tumor cell viability. destroyed outside.

The availability of isolated and purified autocrine polypeptide tumor motility factors This makes it possible to obtain anti-AMF antibodies with specific binding affinity. It takes Antibodies can be either polyclonal or monoclonal and Produced by standard technology routes well known in the art. Such antibodies may also be suitable Can be labeled with radioisotopes or fluorophores and other markers or ligands, It can be used to detect, quantify and/or localize AMF in tissues or body fluids. Change Radiolabeled AMF together with unlabeled AMF was used to measure the amount of AMF receptors. Available in standard competitive examinations. Such binding assays for measuring receptor abundance are: Carry out as follows.

AMF binding assay: Purified AMF is isolated using the standard Bio-Rad enzyme bead method. amount of increase Labeled AMF was added to 100.000 cells in the presence or absence of a 100-fold excess of cold competitor. Culture in a volume of 1 ml with A2058 melanoma cells.

Incubation is carried out for 40 minutes at 37°C and cell-bound radioactivity is separated by centrifugation.

AMF binding shows 80% specific binding and saturation at approximately 30,000 receptors/cell. Shows sum. Scatchard analysis according to the standard method gives an estimate within the range of approximately 0.5 nM. Linear relationship between specifically bound/free ratio and specifically bound AMF in kd shows.

Additionally, detection of cancer in humans is made possible by this discovery and The testing of human samples for this purpose will now be illustrated using a urine sample of

Collects urine samples from bladder cancer patients and has a 10 kilodalton exclusion filter A centrifugal microconcentrator (AMICON) is used. processed urine Reconstitute at 10x concentration in sterile phosphate-buffered saline, pH 7.5, and use. Store at -20°C until cold. Pathologist grade tumor grade using a scale of 1 to 3 To be determined. A grade 1 tumor indicates the largest tumor size. , grade 3 tumors show the least differentiation. American medicine for bladder tumors Staged according to Likan Joint and Committee TNN Classification Ru.

Assay of urine sample: Although any cell line that responds to AMF can be used, a preferred cell line is human MD A435 cells (ATCC). Transfer concentrated urine samples to microwells as described above. Applies to chamber tests. Each sample was tested with an antibody directed against human tumor AMF. A series of dilutions are tested with and without the addition of cartilage. AMF units are inhibited by antibodies. Record the percentage of tumor cells stimulated to migrate by the sample. in general , stimulation migration higher than 80% was suppressed by an antibody concentration of approximately 10 μg/ml. be done.

As shown in Table 3, control urine with non-neoplastic disorders such as kidney stones had a significant amount of of motility factors. All of the bladder mobile cell carcinoma cases have positive movement in the urine showed a sexual response. The highest amount of motility factor production occurs in high-grade tumors or stage D (metastasis) Found in the urine of patients with II tumors.

Table 3 1 Control ks75 5 0.52 Control ks76 9 2 3 Ca spot 32 5 4 Papillary TCC648 5TCC77443 6TCC699814 7TCC7312332 8Recur TCC7913022 9TCCn485 169 14 10 TCC■491 105 8 11 TCCn554 41 12 12 TCCm457 72 6 13 TCCstgD584 234 25TCC-Mobile cell carcinoma of the bladder Recur TCC - Recurrence TCC TCCII-Grade ■ TCCIn-Grade■ TCCstgD-transition TCC KS-kidney stone SE - standard error Of course, antibodies against AMF will block or suppress AMF activity, thereby IL can be used to inhibit tumor invasion or metastatic growth that is dependent on tumor cell movement. . Additionally, the availability of such neutralizing antibodies may be difficult for those affected by conditions such as breast cancer and melanoma. By administering effective amounts of AMF-antibodies to prevent these conditions from developing. This makes it possible to treat conditions such as breast cancer and melanoma. Cure cancer and metastasis The pharmaceutical composition for treating tumor cells contains AMF in an amount effective to inhibit tumor cell movement. A neutralizing antibody to Prepared by pure blending.

The means for detecting tumor aggressiveness and/or metastatic activity may also include (a) AM Antibody with specific binding affinity for F; (b) Labeled AMF: (c) a kit for measuring AMF and/or receptor activity in a body sample; Educational materials and unlabeled materials for conducting tests utilizing labeled antibodies and AMF This is now made possible by a kit consisting of a separate container containing AMF.

Microtiter plates, micropipettes, equipment for reading antibody titers, etc. Accessories are commonly found in such kits and are conveniently included in the kits of the present invention. You can absorb it.

In summary, the present invention opens new avenues for understanding the mechanisms controlling tumor cell invasion. and open new strategies for cancer diagnosis and treatment. Epithelial cells are usually , does not exhibit invasive behavior. The motility factors described here influence normal leukocyte migration. does not affect Therefore, treatments aimed at suppressing the factors according to the invention The drug should have low toxicity to normal quiescent tissues. tumor cells The present invention suppresses invasion of cancer and prevents metastasis from the site to invasive cancer. Pharmacological formulations can be effective cancer-inhibiting agents. Also, metastases grow Since local invasion may be necessary during tumor invasion, inhibitors of tumor invasion are , the growth of established metastases can be prevented. Additionally, such agents may May inhibit tube formation. Antibodies against motility factors or their receptors are applied by tissue immunohistochemistry, radiosynthesis, or serum immunoassay can be used to localize metastases and predict cancer aggressiveness in individual patients.

As gene products, autocrine motility factors or their receptors are a new class of Define oncogenes. The expression levels of these genes in patient tumors are similar to those in body samples. Monitoring the amount of AMF in patients may provide important diagnostic information.

Of course, invasion and metastasis are the main causes of cancer treatment failure. The present invention provides (a) preliminary detecting invasive lesions and preventing their progression; (b) correcting the aggressiveness of the patient's tumor; (C) new clinical strategies to identify and eradicate micrometastases; provide an overview. One of the least understood aspects of tumor invasion is tumor cell migration. Ru. The present invention allows determination of the role of tumor cell motility factors.

The examples and embodiments described herein are for illustrative purposes only, and Various modifications or changes may be suggested to those skilled in the art and are within the spirit and spirit of this application. and is to be encompassed within the scope of the authority and appended claims. Ru.

Engraving (no changes to the content) Fig, 1 AMF Suyaya'n Yard AMFCL stand (nM)

Claims (10)

[Claims] 1. Stimulates random migration of productive cells, has a molecular weight >30,000 and lasts for 100 days. an isolated substantially pure mammalian product characterized by being inhibited by cough toxins Cell polypeptide. 2. At least in part: [There is an array] 2. The polypeptide according to claim 1, having an amino acid sequence order of at the NH2 terminus. 3. An antibody having specific binding affinity for the polypeptide according to claim 1. 4. Administering an effective amount of the antibody according to claim 3 to a host suspected of suffering from a malignant tumor. A method for inhibiting metastatic growth characterized by inhibiting tumor growth. 5. The method according to claim 4, wherein the malignant tumor is melanoma, breast cancer and bladder cancer. . 6. An antibody with specific binding affinity for autocrine motility factor (AMF) tumorigenic or metastatic activity in the body characterized by comprising a container containing Kit for detecting sex. 7. Separately (a) labeled AMF; (b) unlabeled AMF; and (c) AMF-accepting A container containing instructions for carrying out tests on body samples to determine the level of body activity. 1. A kit for measuring the amount of AMF cell receptor, characterized by comprising a device. 8. Reacting human samples from patients suspected of having cancer with AMF-sensitive cell lines and measuring the motility induced in a sensitive cell line by said body fluid. A method for detecting the presence of cancer in humans based on its symptoms. 9. suppressing the motility induced by the human body sample with an anti-AMF antibody; The method according to claim 8. 10. 9. The method of claim 8, wherein the cancer is human bladder cancer, breast cancer or lung cancer. .