JPH03503763A - Common Cancer-Related SCM-Recognition Factors, Preparations and Methods of Use - 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] °-Common Cancer-Related SCM-Recognition Factors.

Preparation products and methods of use Technical field This invention relates to a general SOM-recognition factor related to cancer, the SCM-recognition factor Method of using factors in 80M test, method of image diagnosis of cancer cells, directing anticancer drugs to cancer cells Regarding the orientation method 1, etc.

Background technology Many diseases that occur in humans and animals are particularly related to diseases or conditions present in the blood. It can be detected by separately related foreign substances. The consequences of such diseases and Testing of antigens or other such substances produced by It is extremely promising as a diagnostic tool for early detection and treatment of certain diseases. It takes Detection methods for substances are required to constitute a practical diagnostic method for health care providers. It must be reliable, reproducible, and sensitive.

Such methods can be carried out by persons of ordinary skill and trained in research methods. must be able to be implemented quickly and at low cost.

For example, it is used to treat various malignant tumors that afflict humans and animals, commonly referred to as cancer. especially since most treatments are only effective in the relatively early stages of the disease. Early detection is the key to effective treatment. For example, many are toxic to malignant tumor cells. Chemotherapy drugs are also toxic to normal cells and may be used to treat cancer or further develop cancer. The high doses required to inhibit cancer progression in some cases lead to discomfort and serious side effects. Also Surgery is most often only effective before the disease spreads or metastasizes. extremely Many cancer cases are only reported too late for effective treatment. .

Therefore, there is a strong need for reliable tests that can diagnose cancer at an early stage. A great deal of research has been done on this subject. In this regard, early diagnosis of cancer New tests and methods are being developed to

One of the highly desirable features of such tests is that, depending on the materials used, all tests The ability to detect type of cancer in general or to detect a specific type of cancer. . The former use of such tests is for large patient It is extremely important in population screening. Such mass screening In research, tests that depend on a specific type of cancer are difficult to test because there are literally hundreds of types of cancer. Tests that are undesirable and can only detect one or a few diseases are Many cases of cancer are extremely easy to overlook. Generally, these patients Does the patient have symptoms that cannot be easily linked to cancer?

or exhibit unclear systemic symptoms, so a specific type is suspected and identified. There are no standards for testing.

On the other hand, once the presence of a malignant tumor is known or strongly suspected, certain types of It is desirable to have a test that can accurately determine malignancy. Such tests Many of the most effective cancer treatments, such as therapy, are for one type of cancer or at most malignant cases. It contributes significantly to effective treatment. Therefore, the potential clinical effect have

Meeting this demand and improving the diagnosis and early detection of cancer in humans and animals In the research.

Cell shape of lymphocytes that survive when exposed to phytohemagglutinin or cancer-associated antigens Structuredness of the quality matrix ytohlasmic Matrjx (SCM)) Test methods have been developed. This method is described in the following publications: L, 0ercek, B, 0ercek, and C, 1, V, Frank lin.

“Biophy*1cal Differentiation Between n LympOCytesfrom Healthy Donors, Pa patients with MaligontDiseases and 0th er　Disorders,　Br1t,　J,0ancer“Applica tion of the Phenomenon of Changes 1n the 5 trueness of Cytoplasmic Ma trix (SCM) in the Diagnosis of Maligan t　Disorders:　a　Review”.

By this method, a subdivided population of nonpaocytes potentially responsive to SCM was tested. A patient's blood sample is separated from the blood sample to identify malignant tumor tissue or malignant tissue. Extract tumor tissue. If the blood sample donor is suffering from crab malignancy, phosphoryl from a donor not suffering from malignancy (characteristics distinguishable from bulbar SCM reactions). There is a characteristic SCM reaction. What is the SCM reaction?

Changes in the intercellular fluorescein polarization action of phosphorus cells in response to SCM determined by measuring the

The changes seen in the 80M test are thought to reflect changes in the internal structure of the ball. It will be done. These oddities.

When polarized light is used to excite fluorescein present in cells, the polarized light of the cell Expressed as a decrease (decrease) in action. Fluorescence polarization is a measure of intercellular oka11 Therefore, the greater the mobility between cells, the smaller the determined fluorescence polarization effect. It becomes. From changes in the conformation of the organelles that produce the observed fluorescence polarization This is considered to occur. Mitochondrial changes are due to changes in the membrane of midcosidria. It is thought to result from contraction of the te (intimal fold). SCM is a polymer and water molecules , between small molecules such as ions, adenosine triphosphates, and adenosine cyclic phosphates. reflects the power of interaction. Perturbations in these interactions result in changes in the SOM.

The 80M test is capable of responding to relatively small amounts of malignant cells. He weighs about 70 yen. Approximately 10 cells in the 80M test are sufficient to detect lymphocytes in a malignant tumor-specific pattern. Make it react. In mice, 7.5xlO and a small amount of Ehrlich ascites (tumor ) When the cells are transplanted, the reaction pattern in the 80M test changes #): Specific to cancer responses to antigens are induced, while normal responses to PHA are virtually eliminated. (L, 0ercek & B.

0ercek, “Changes in SCM-Responses” f Lymphocytesin Mice After Implantat ion with Ehrlich AscitesSCM test, blood sample Compared to traditional methods, it causes relatively little discomfort to the patient except for the collection of It is also possible to detect cancer at an extremely early stage.

However, this method has drawbacks. for example.

Prepare crude extracts from cancer tissues, etc., or use the cancer tissues themselves as sources of cancer-related antigens. need to be used.

There are several problems with the use of cancer tissue or extracts of cancer tissue in the 80M test.

Obtaining the required amount of tissue may be difficult. Use of whole tissues or crude tissue extracts use introduces interfering substances into the testing process. These interfering substances adversely affect the sensitivity of the test. or adversely affect the test results themselves. Presence or absence of these interfering substances can easily vary from batch to batch of cancer tissue.

80M results in deleterious variations in the test. Additionally, interfering substances may be present in whole tissues or crude extracts. Because of their existence, their identification or quantification is extremely difficult.

Therefore, we have identified factors that elicit responses by lymphocytes that respond to SOM. It is highly desirable to read and purify it. The use of such purified factors may result in interference To promote SOM cancer screening tests and aid cancer research since there is no become.

Disclosure of invention We use donor blood in animal and human body fluids, specifically plasma and urine. Cancer-related extracts and/or or in lymphocytes reacting to the same SCM as lymphocytes reacting to cancer tissue. We have discovered a cancer recognition factor that gives a response. As described herein, These factors are in the singular, ``Common Cancer-Related SCM-Recognition Factor J,'' sO M-recognition factor" or simply rscM factor j.

Factor 80M is a peptide or peptide-like substance that is closely related to 2.3. It is thought to consist of peptides. The 80M factor has the sequence Phe-R1-Lys-P ro-Phe -R1-Phe-Rs -Met-R4-R4-R4-Rt (However, R1 is selected from the group consisting of Asn and Gin, and R2, R1 and R number are selected from the group consisting of Asn and Gin. R6 and R7 are each independently selected from the group consisting of Va l, Leu, and Ile. are independently selected from the group consisting of Asn and Gin]. It is thought to contain acid fragments.

This sequence does not include the amino acid terminus of factor 80M.

A common cancer-associated SOM-recognition factor is one with a nominal molecular weight of 1000 daltons. Passed through a filter with a nominal molecular weight cutoff of 500 daltons. A low molecular weight peptide retained in the filter, and the peptide is a standard 8 isolated from cancer patients when used to stimulate lymphocytes in the 0M study At least a 10% decrease in intercellular fluorescence polarization of SOM-reactive lymphocytes. give.

The factor purified by the method described below has approximately the following amino acid composition: : (Asx1.Glxx.

8er, His, GlyssThrsArg, Alam, Tyr, Met, Va lx.

Phem, IIe, Leua*Lysm).

Based on the amino acid sequence of the tryptic fragment of factor 80M, one preparation of the factor The 16 amino acid segment within has the amino acid sequence Phe-Asn-L y　s　-Pr.

-Phe-Val-Phe-Leu-Met-11e-Asp-Gin-Asn -Phe-8er-Lys. And that segment is the factor amino acid Does not include the end.

Additionally, a 14 amino acid segment within another preparation of factor 80M has an amino acid sequence of Phe Asn-Lys-Pro-Phe-Val-Phe-Leu-Me t-11e-Asp-Gln-Asn-Thr-Lys'. and That segment also does not include the amino acid terminus of the factor.

Both of these two tryptic fragments are fully active in the 80M test. two The first 13 amino acids, which are common to all passages, are the most important for the generation of SOM-activity. be. Therefore, another important aspect of the present invention is that the peptides consist only of peptides of defined sequences. 80M factor, and these peptides can be directly added to water as having 80M activity. or may be considered to have such activity. Most broadly, such factors is a peptide having all at least 13 amino acid residues containing essentially the following sequence: Consisting of: Ph e -R4-Lys -Pr o-Phe -Rq -Phe -FLs -Me t -R4-R4-R4-R-r (However, % R1 is selected from the group consisting of Aan and Gln, and R8, R, and R numbers are each independently selected. va'*Leu m and Ile, and RII is Asp. and Glu, and R6, R, and F are independently selected from the group consisting of Asn and Glu. Select from the group consisting of n]. It is desired that this factor essentially contains one peptide. Yes.

When utilizing the complete sequence information from two tryptic fragments, it is assumed that each The origin of the 80M factor of two different parallel sequences containing the 13 amino acid fragments of It is.

The first class is most broadly 1 essentially Phe-Rt-Lys-Pro-Phe -R4-Phe-Ra -Me t-R4-Rs -FLs -RnR,BL Array of ys [However, R1 to R? is defined before, Ra is Ser and Thr [selected from the group consisting of] only peptides having at least 15 amino acid residues] It will be. Preferably, this factor consists essentially of only one peptide.

More narrowly defined factors within this class are essentially Phe-Asn-Ly s-Pro-Phe-Va I-Phe-LeuMet-11e-Asp- It consists only of peptides containing the sequence Gin-Asn-Thr-Lys. these The peptide may further include flanking sequences on either side of the specified sequence. this Desirably, the agent comprises essentially only one peptide.

The most narrowly defined factors in this class are essentially Phe-Asn-Lys -Pro-Phe -Va I-Phe-Leu-Met-11e-Asp-G It consists of an essentially pure peptide consisting of lu-Asn-Thr-Lys.

The second class is 1 essentially the most widespread including Phe-Rs-Lys-Pro- Phe-R1-Phe-R1-Me t-L -R4-R4-k -Phe-R s - Lys C However, Rt ~ R, l are defined previously, R8 is Ser and Th at least 16 amino acid residues comprising the sequence selected from the group consisting of Consists only of peptides. It is desired that this factor essentially contains only one peptide. Delicious.

A more narrowly defined factor within this class is the essential K　Phe-Asn-Ly s-Pro-Phe-Va I-Phe-Leu-Met-11e-Asp It consists only of peptides containing the sequence Gin-Aiu-Phe-Thr-Lys.

Konera's peptide contains a specific sequence of ICs on both sides and flanking sequences. I can see it. Preferably, this factor consists essentially of only one peptide.

The most narrowly defined factor in this class is the essential K　Phe-Asn-Lys -Pro-Phe -Va l-Phe-Leu-Met-11e-Asp-G Essentially pure compound consisting of amino acids in-Asn-Phe-Thr-Lys Consists of petite. This peptide is a SOM-reactive protein from a donor suffering from cancer. 5 x 10 femtograms of peptide when used to stimulate lymphocytes ( activity in the 80M test such that fP) gives a decrease in polarization value of at least 30 degrees. It is desirable to have sex.

The 80M factor consists of molecules with an apparent molecular weight of less than 1000 Daltons. body fluid consisting of molecules with an apparent molecular weight of 1000 Daltons or more from both sides of the body. External treatment of body fluid from a donor suffering from cancer to isolate fractions of It can be generated by Its body fluids consist of peripheral blood, urine and plasma Choose from the group. After ultrafiltration, the factor is purified with each step resulting in a more highly purified factor. It is further subjected to a purification process consisting of several steps.

The first step in this purification process is to use an ultrafilter with a fractionation range of θ to about 700 daltons. The factor consists of elution from a gel filtration column that can separate salts from Elutes at about 0.3 to 0.5 times the total romatograph bed volume.

The second stage Iv is a gel filter with a fractionation range of approximately 1500 to 30,000 l/'. The elution factor of the overcolumn is approximately 0.4-0.0% of the total chromatographic bed volume. Elute from the column at 6.

The next step in this purification process is about 0.28M to 0.31M ammonia bicarbonate. Elution from an anion exchange column of dimethylamine ethyl cellulose with It will be.

The final step is to refine the factors to substantial homogeneity by reverse-phase high-pressure liquid chromatography. It consists of manufacturing.

It may be desirable to use a more highly purified preparation of the factor in the 80M test. However, factors from all stages of purification, including the initial extra-P solution, can be used in the test. Wear.

80M factor is one type regardless of the type of cancer present in the donor from which the 80M factor is isolated. Potentially responsive to SCM obtained from donors with different types of malignancies Gives a positive reaction to lymphocytes. Hence the term "general". The factor is malignant tumor Little or no 8CM reaction was induced in the Rinba spheres when derived from non-containing donors. do not. The ability to act as a general cancer-associated antigen makes the factor sensitive to the presence of malignant tumors. Makes it useful for general screening of blood samples.

The factor also has other interesting properties. One of these properties is the absence of malignancy S of lymphocytes that react to SCM when the factor comes into contact with lymphocytes obtained from a donor. properties that improve the CM response, whereby lymphocytes It reacts with cancer-related antigens, but does not react with mitogens (mitogens) after contact. do not have. A second property is that the line of human bone marrow cells potentially It has the property of lowering at least 90 inches in the 62 direction.

Another aspect of the invention is a method of using 80M factors associated with common cancers in the 80M test. Regarding. Most commonly, this method involves (1) lymphocytes from a mammalian donor and general (2) Suspension of lymphocytes. Reduced degree of organization of the cytoplasmic matrix of lymphocytes obtained from the fluid contacting process Testing lymphocytes from mammalian donors by determining the presence of malignancy or non-existence.

A preferred method to quantify the decrease in degree of organization is il) General cancer-related SOM-recognition Measuring the fluorescence polarization Ps on an aliquot of lymphocytes contacted with the factor: ( 2) Measure fluorescence polarization Pc on an aliquot of control lymphocytes that were not contacted. Step: and (31P　　　　　Contains the step of determining PcO ratio. Approximately 0.9 or less The Ps/Pc ratio indicates a positive response to factor 80M and a malignant tumor in the lymphocyte donor. Indicates the presence of a tumor.

A further preferred method is to use Ps and another constant of lymphocytes contacted with the mitogen. The SOM reaction ratio RR3CM is determined by comparing the amount of fluorescent polarized light PM. : RRscM=P S/PM A R of about 0.9 or less indicates malignant CM in the lymphocyte donor. Indicates the presence of a tumor.

Apart from this, methods of using the 30M factor in the 80M test include (1) blood sample Step of separating lymphocytes potentially reactive to SCM from: (2) isolated lymphocytes; Stimulate lymphocytes by contacting lymphocytes with common cancer-associated SOM-recognition factors. Stimulating step: (3) The stimulated lymphocytes undergo intracellular addition to the fluorogenic compound. contacting a fluorogen precursor, defined as a water-degradable, non-fluorescent compound; , by infiltrating lymphocytes with fluorogenic compounds for intracellular enzymatic hydrolysis. Step of producing stimulated fluorescent-containing lymphocytes: (4) Stimulated fluorescent-containing lymphocytes (5) Exciting lymphocytes with polarized light to cause them to fluoresce; Polarized fluorescent light emitted from fluorescent lymphocytes in vertical and horizontal directions (6) determining the polarization value of the lymphocytes that generate fluorescence by measuring; and (6) Determined polarization values of lymphocytes containing fluorescent light and control phosphorus of the same donor volume. The presence of cancer in the lymphocyte donor's body can be determined by comparing the polarization value of the lymphocytes with the lymphocyte polarization value. It consists of a process of indicating whether or not. Steps (3) and (4) can be performed simultaneously.

Lymphocytes can be excited with vertically polarized light. Using vertical polarization The polarization value of IH is the intensity of polarized fluorescence in the vertical and horizontal planes, respectively: G is Correction factor for the unequal transmission of horizontal and vertical components of polarized light through the optics of a spectrophotometer It is a number.

The present invention not only provides a diagnostic method for identifying subjects suffering from cancer, but also It also includes the patient's treatment method. These methods utilize common cancer-associated SOM-recognition factors. The observation that the in vitro cytotoxicity of lymphocytes towards cancer cells Based on.

Based on these observations, a decrease in the in vivo activity of SOM-recognition factors may be associated with cancer cells. Immunology with lymphocytes should increase the efficiency of surveillance.

Most commonly, this treatment involves at least one of the body fluids being diagnosed with common cancer-related SOM. This is for patients who include certain factors. The treatment is (1) s OM-recognition factor In vivo release of the factor by treating body fluids containing it to selectively inactivate it. Step of reducing the effect: (2) Returning the body fluid to the patient to reduce the patient's resistance to the cancer. It consists of a process of increasing.

One use of this general size is to treat one body fluid, one body fluid is peripheral blood, and one body fluid is peripheral blood. The process involves dialyzing the peripheral blood to obtain peptides with an apparent molecular weight of less than 1000 Daltons. by removing the tide and thereby selectively inactivating the SCM-recognition factor. Become.

Another application of this general method for body fluid processing techniques is to identify SCM-recognized agents in body fluids. This consists in neutralizing the antibodies with antibodies specific for the factor or with Fab fragments of said antibodies.

The Fab fragment is capable of monovalent binding to the SCM-recognition factor.

Furthermore, the present invention also includes a method of imaging (image diagnosis) cancer cells. this The method is (1) using an antibody against a common cancer-related SOM-recognition factor as an imaging substance. and (2) image diagnosis of cancer cells using the labeled antibody. The process consists of

The present invention further provides (1) using antibodies against common cancer-related SOM-recognition factors as anti-cancer drugs. Targeting anti-cancer substances to cancer cells by labeling them with The method consists of administering to a cancer patient a labeled antibody that is capable of binding to cancer cells. anti cancer Includes methods of directing substances to cancer cells.

Furthermore, the present invention provides a method for detecting common cancer-associated SCM-recognizing factors in immunoassays. Determining levels of common cancer-associated SOM-recognition factors in body fluids using antibodies Includes methods. Immunoassay methods include radioimmunoassay, fluorescence immunoassay, and enzyme-linked immunoassay. It can be an immunosorbent assay or a precipitation immunoassay, such as a turbidimetric assay. Ru.

These and other features and advantages of the invention will be found in the following description and claims. You will be able to understand it even better.

Definition of a number of terms used repeatedly in the following discussion, examples, and claims. Collect information for convenience.

"General" refers to 1 the donor of the body fluid from which the 30M factor of the invention is purified, or the 80M factor of the present invention. Cancer afflicting the donor of the lymphocytes used with factor 80M in the M trial means that it is non-specific with respect to a particular type of

Non-fluorogenic compounds that are raised and converted into fluorogenic compounds within the cell by hydrolysis An example of the compound used herein is fluorescein diacetate (FDA ).

“Standard 80M Test J: 6X10 cells/- with 1.01 lymphocyte suspension and 0. 1- mitogen or antigen and FDA'' as a fluorogen precursor. .

Use an excitation wavelength of 470 nm and an emission wavelength of 510 nm for fluorescence polarization measurements. 80M test.

Both of these terms mean that the separation of molecules by ultrafiltration according to size is by a factor of 80M. Approximate molecules in a range of sizes and depend on conformation and size. It can be separated from molecules with molecular weight.

A unique product that exhibits 80M activity and contains all proteins and large peptides. a pure state in which the vast majority of other molecules with physical activity are not present material.

A proteolytic enzyme that cleaves the peptide chain after a lysine or arginine residue A peptide that has been cleaved from a larger peptide by the action of an element.

Best mode for carrying out the entire invention The present invention relates to the discovery of peptides that are common cancer-associated scM-recognition factors and The present invention relates to purifying peptides to substantially uniformity. The factor is 1000 Daltons nominal Passes through a filter with a molecular weight cutoff, but with a nominal molecular weight of 500 daltons. is retained by a filter with a quantity cutoff. The factor is.

(Asx1.Gig, Ser, His, Glyg, Thr, Arg.

Alas, Tyr, Met, ValB%Phes, Ile%Leu@.

LysI) has an approximate amino acid composition. The two active tryptic fragments are factor It has been isolated from another specimen. These fragments contain the amino acid terminus of the first factor molecule. do not have. The first of these fragments is Phe-Asn-Lys-Pro-Phe-Va I-Phe-Leu-Met-I1e-Asp-Gln-Asn-Th It has 15 amino acids with the sequence r-Lys. Kone, the second of the fragments is P he-Asn-Lys-Pro　-Phe-Val-Phe-Leu-Met- Has the sequence 11e-Asp-Gin-Asn-Phe-8e r-Lys It has 16 amino acids.

The nature of the factor is that it completely modifies the SCM response of donor lymphocytes without malignant tumors and Ability to reduce in vitro cytotoxicity of lymphocytes to malignant tumor cells as well as donor Some of the cross-reactivity of factors isolated from cancer with many different types of cancer are listed below. It will be posted.

Properties of 80M factor Amino acid set of common cancer-associated SOM-recognition factors purified from plasma of breast cancer patients decided to become The results are shown in Table 1. RP-) purified by IPLC Freeze-dried samples of the factors were dissolved in 6N HO4 in sealed glass ampoules under a nitrogen atmosphere. Hydrolyzed with 2% glycolic acid at 110°C for 24 hours.

After hydrolysis, the samples were lyophilized. Residue is 0.04M sodium borate buffer It was dissolved with 2% sodium dodecyl sulfate in the solution. Amino acids are listed in the primary publication. After separation by HPLO, O-phthaldialdehyde is detected by their fluorescence. Determined as a derivative: B, N. Jones, S., Paabo and S,5lein,”Am1n.

Ac1d Analysis and Enzymatic 5equence Determination of Peptide by an Impr oved 54-PhthaldialdehydePrecolumn L abeling Procedure, “J, Liquid Ohroma t.

Analysis by HPLC”, Altex Division, B eckman Instruments, Inc., Berkeley, C. a1ifornia　94710゜Table 1 Relative molar amino acid composition of S0M factor Amino acid Relative molar assembly As x 2.10Glx 3.20Set 1.27His 0,65Gl)1 4.60Thr 1. 11Arg 0.86Ala 　 　 　 z60Tyr 　 　 　 　 　 　 1.58 Val 3.2 2Phe 3.0711e O, 86 Leu 2.87 Amino acid composition is nitrogen Dry with 2% thioglycolic acid in 6N hot in a sealed glass ampoule under an elementary atmosphere. The dried peptides were determined after hydrolysis. After hydrolysis, the sample was dried for 1 yen and the remaining 2% SDS buffer in 0.04 M sodium borate. Ami No acids are used as dialdehyde derivatives according to Beckman Instruments specifications. Decided. Amino acids Cys and Pro were not determined.

The amino acid composition data in Table 1 indicates that the active agent is a peptide. that day ta is the approximate amino acid part g (Asg, Glxm, Ser, His, Glg, Th r, Arg.

AIam, Tyr, Metl, Valm, Phea, Ile, Leum, Ly The best match was with the peptide with sm).

The amino acid sequences of segments isolated from two different samples of factor 80M are In-line 120APTH - Protein Sequencing Device Combined with Amino Acid Analyzer Automatic Edman minutes using (App ed Biosystem 477 human type) It was determined by solving the problem. 2.3 types of cancer 1 such as breast cancer, lung cancer, large Intestinal cancer.

Whole SOM factors isolated from patients with bronchial cancer or cervical cancer were subjected to Edman degradation method. Time 1: No reaction occurred.

It was found that the amino acid terminus of factor 80M was blocked. Therefore, the molecule's To determine the sequence of a fragment, a flag with the amino acid at the free amino acid end must be used. I needed to get some ment. Such fragments are prepared by trypsin digestion followed by triplication by reversed-phase high-pressure liquid chromatography as detailed in the Examples below. The factor was obtained from another purified sample by purification of the tocin fragment. These hula one from factor samples from the plasma of lung cancer patients, and the other from blood from breast cancer patients. Obtained from serum.

Specific fragments in each case (later shown to have 80M activity) is 30.4 volume chips of phase A and 69.6 volume chips of phase B in a total volume of about 30 μt. It was eluted with For sequencing, the seven fragments of SCM-active tryptosin were The generated PTs were directly interspersed on the disk of the column determiner, followed by the Edman decomposition method. Identification of H-amino acids in a coupled online PTH-amino acid analysis instrument Computer-managed automated sequencing analysis was performed by sequential cycles of

The sequencer's sequence calling computer program was isolated from the plasma of lung cancer patients. We identified 16 amino acid sequences as the sequences of 7 fragments of factor 80M. So The sequence is Phe-Asn-Lys-Pro-f'he-VaI-Phe- Leu-Me t-Asp-Gl n-Asn-Phe-8e r-Lys It was. Similarly, a computer program generates a sample of factors isolated from the plasma of breast cancer patients. A 15 amino acid sequence was identified as the fragment sequence. The sequence is Phe -Asn-Lys-Pro-Phe -Va 1-Phe-Leu -Met -I1e-Asp-Gln-Asn-Thr-Lys.

The first 13 amino acids in these two trypsin fragments are identical. It is one. Although their carboxy termini differ, one missing amino acid P If we insert he into the shorter fragment.

The only difference is the substitution of Thr for Set, a highly conservative substitution in the peptide 80M factor, which has been purified so as not to affect the function of The IJSCM study showed that it was used as a stimulant for lymphocytes isolated from patients with fully active in This activity was determined by the assay method used during the purification of the factor. It can be shown as follows. Details of the results of the assay are shown in the Examples below. best life The quality is obtained in the final purification step, reversed-phase high-pressure liquid chromatography.

Fraction 1/10m of O containing 40 μM of peptide was determined from cancer patients. When used to stimulate SOM-reactive lymphocytes isolated from 6 cells, but stimulated the same subfraction of lymphocytes isolated from healthy donors. The intracellular fluorescence polarization effect was not reduced.

sex 15 amino acid trypto cleaved from factor 80M isolated from plasma of breast cancer patients Synheptide fragments (breast fragments) and i 16 amino acids tryptosine peptide fluorinated from isolated factor 80M Both lung fragments were completely tested in the 80M test. X10 P, or about 16,000 molecules).

In the 80M study, it showed complete activity when used as a stimulant for lymphocytes in cancer patients. showed his sexuality. Lung fragments are equally well represented in lymphocytes from lung and breast cancer patients. 80M test when used to stimulate lymphocytes from normal donors. I didn't react.

Both lung and breast fragments were fully active in the 80M test. Because they share only the first 13 amino acids of their sequences.

This common sequence of 13 amino acids is the most important sequence in the generation of 80M activity. be. Therefore, the sequence of only these 13 amino acids, called "core", Phe -Asn-Lys-Pro-Phe -Va l-Phe-Leu-Met-1 1e-Asp-Gin-Asn is believed to have 80M activity.

This sequence is the only sequence of 13 amino acids thought to have 80M activity. do not have. Certain amino acid substitutions, called “conservative” amino acid substitutions, are What is often done in peptides without changing conformation or function is It is a well-established principle of protein and peptide chemistry. In the array of cores Changes affecting such amino acids a, Ile and Le for Val u and vice versa.

Asp to Glu and vice versa and Asn to Gin and vice versa including. Therefore, the core structure in which any or all of the above conservative amino acid substitutions are made It is believed that an unusual peptide molecule with a structure exhibits 80M activity and therefore It is considered to be a part of it.

Lung and Breast Fragments are Both Larger Common Cancer-Related 80M-Recognition Molecules isolated as part of Such fragments can be incorporated into larger molecules''1 It is clear that their 80M activity can be retained when n. Therefore, the above Incorporates a 13 amino acid core sequence with or without conservative amino acid substitutions It is expected that large peptides will also exhibit 80M activity.

The occurrence of these two types of modifications of the core sequence provides yet another important aspect of the invention. I can do it. This is an SCM consisting of only peptides with a defined sequence = active factor; Those peptides have been directly hydrated to have 80M activity or It is thought that it has. Such factors most broadly include substantially Phe-R1-Ly s-Pro-Phe-R4-Phe-R1-Met-几4-Rs-R6-R? (However, %RI is selected from the group consisting of Asn and Gln, and R1, R, and R4 are Each independently selected from the group consisting of Va1%Leu and Ile, and R is Asp. , and Glu, R6 and R? are Asn and Glu independently? a peptide having at least 13 amino acid residues comprising the sequence selected from the group consisting of Consists only of chido. Preferably, this factor contains substantially only one of the peptides. stomach.

When complete sequence information from lung and breast fragments is utilized, it is This results in an 80M factor of different parallel laths. and each Contains sequences derived from the sequence by conservative substitutions. These long peptides One further possibility for each conservative amino acid substitution, namely the replacement of Ser. of Thr and vice versa. The equivalent of these amino acids is that the lung fragment is S Although it is et.

The breast fragment is indicated by having Thr. These amino acids The position in each of the 7 fragments is the deletion of Phe at position 14 of lung fragment F. You can align them by doing this.

The first class of factors is the sequence of the breast fragment, Phe-Asn-Ly. s-Pro-Phe-Va 1-Phe-Leu-Met-11e-Asp- Derived from Gin-Asn-Thr-Lys. This class most commonly includes Qualitatively Phe -R1-Lys-Pro-Phe-1-Ph e -FLs -Me t -R4-R11-R4-R, 7-R6-Lys containing sequence It consists only of peptides having at least 15 amino acid residues. In this array, , Rs to R are defined above and possible amino acid placements in the core sequence. R8 is selected from the group consisting of Ser and Thr. This factor is essentially Within this first class, it is desirable to include one peptide and to further specifically limit the The factor is substantially Phe-Asn-Lys-Pro-Phe-VaI Phe-Leu-Met-11e-Asp-Gin-Asn-Thr-Lys Consists only of peptides containing the sequence. This is the original sequence of the breast fragment.

These peptides contain additional flanking sequences on either side of the specific sequence be able to. These flanking sequences can then be of hollow length.

Desirably, this factor also contains substantially only one peptide.

The most narrowly defined factor within this first class is.

Essentially PRe-Aan-Lys-Pro-Phe-Val-Phe- Leu-Me t-I Ie-Asp-Gl n-Asn-Thr-Lys Consists of substantially pure peptides consisting of amino acid sequences. This is flanking array This is the exact sequence of the breast fragment that does not have .

The second class of these factors is the sequence of lung fragments, Phe-Asn-Lys -Pro-Phe　-Va　-Phe-LeuMe　-I　Ie-Asp- It is derived from Gln-Asn-Phe-8er-Lys. This class is the Mohiro is essentially Phe-R4-Lys-Pro-Phe-R1-Phe- Rs-Met R4-Rs-Rs-Rtr-Phe-Rg-Lys It consists only of peptides having at least 16 amino acid residues containing the sequence.

In this sequence, Rs~R-r are defined previously, and in the core sequence R8 is selected from the group consisting of Ser and Thr, resulting in a possible amino acid substitution. child The factor contains substantially [preferably, only one peptide].

More specifically defined agents within this second class are substantially Phe-As n-Lys-Pro-Phe-Va 1-Phe-Leu-Met-11e- Only peptides containing the sequence Asp-Gln-Asn-Phe-8er-Ly　s Consisting of These peptides rank an additional 7 on either side of a particular sequence. Can contain arrays. and these flanking sequences are of intermediate length Can be done.

Preferably, this factor also contains a peptide with only one substantial IC.

The most narrowly defined factors within this second class are:

Essentially Phe-Asr+-Lys-Pro-Phe-Va 1-Phe- Leu-Met-11e-Asp-Gin-Asn-Phe-8er-Lys Consists of a substantially pure peptide consisting of an amino acid sequence. This is the flanking arrangement. Accurate arrangement of lung fragments without columns.

These factors are expected to have a total activity of <80M and are within the scope of the present invention. It is within the surrounding area.

5, 8 Cross-reactivity of CM factors Lymphocytes isolated from donors with all types of cancer were tested in the 80M study. The factor is defined as a common cancer-related 80M factor. Ru. What type of cancer affects the lymphocyte donor?

It is not necessary that the type of cancer affecting the donor of the body fluid from which Factor 80M is purified is the same.

As discussed below.

Purification of factors from plasma obtained from patients with 19 different types of cancer was always performed using reverse-phase high-pressure liquid chromatography. This resulted in factors eluting at the same position in the chromatography. This is the same molecule Alternatively, this suggests that similar molecules were isolated from all of these patients. actually , Trypsin Fluorase from factor 80M isolated from plasma of lung and breast cancer patients. The amino acid sequence of the fragment is identical to the first 13 amino acids of the fragment. It is shown that. The only difference between the two sequences is at position 14 of the lung fragment. Deletion of a feralanine residue and threonine for serine at the next position This is a conservative substitution. For more information on demonstrating the cross-reactivity of Factor 80M, see below. Examples.

6. Modification of SCM response by common cancer-associated 80M factors -ff-like cancer-associated 80M factor is potentially anti-SCM obtained from cancer-free donors. The ability to modify the response of corresponding lymphocytes by bringing them into contact with the factor. have quality. Prior to contact, the lymphocytes were mitogen-treated in the 80M test. It reacts only with cancer-related antigens and does not react with cancer-related antigens. however.

After long-term contact with factor 80M, the SCM response of cells is modified to produce cancer-related anti-inflammatory agents. It reacts only with atomic agents and does not react with mitogens. In other words, the 80M factor and the Contact with lymphocytes may reduce their response in the 80M test to malignancy-free donors. seen in lymphocytes from cancer-affected donors from the normal reaction of lymphocytes from Turn it into a reaction. Demonstration of modification of the SCM response of lymphocytes from cancer-free donors is as follows. This will be explained in detail in the "Example" section.

Factor 80M potentially inhibits the in vitro innate cytotoxicity of lymphocytes that respond to SCM. The property of significantly lowering the vine. Based on this property, a common cancer-associated ScM recognition factor The molecular molecules participate in the defense of cancer cells against the attack of killer lymphocytes, causing cancer cells to become uncontrollable. It is thought to help limit growth. The normal function of the immune system in suppressing the growth of cancer cells The importance lies in the fact that abnormal forms of cancer often occur in patients receiving immunosuppression. Therefore, it is shown. An important example of this is suffering from acquired immunodeficiency syndrome (AIDS). Kaposi's is a cancer that usually spreads rather slowly in patients. It is the occurrence of an aggressive form of sarcoma.

Common cancer-associated factor 80M modifies the SOM response of lymphocytes from healthy donors selectively reduces the natural cytotoxicity of killer lymphocytes to bone marrow cells. by inactivating the SOM-recognizing factor and subsequently returning the body fluid containing the SOM-recognizing factor to the patient. Therefore, body fluids containing SCM-recognized factors can be treated to reduce the in vivo effects of the factors. thought to be able to be used in cancer management to increase natural resistance to disease. It will be done.

Body fluid power: In the case of peripheral blood, the body fluid treatment process involves dialysis of peripheral blood for 1000 Daltons. The appearance of the following steps consists of removing molecular weight peptides. Aside from this.

The process of processing the body fluids may be carried out in such a way that the agent is isolated from antibodies specific for it, or fragments of said antibodies, e.g. It consists of neutralization with Fab (antibody-binding) fragments. The Fab flag The ment is monovalent so that each fragment molecule binds only one molecule of the factor. Can be combined. this is.

If the formation of large factor-antibody complexes is undesirable, complete anti-antibody Use of the body is preferred.

The use of this general cancer-associated SOM cancer recognition factor is detailed below.

The first step in the purification of factor 80M is to extract ultra-f fluid from the body fluids of cancer-affected donors. It is to obtain.

The body fluid can be peripheral blood, plasma or urine; if the body fluid is peripheral blood, The blood must be centrifuged to separate red blood cells from plasma.

The donor of the body fluid used for isolation of factor 80M is the lymphocyte used in the 80M test. They can be the same or different with respect to each other.

Limit - In the past, body fluids consisting of molecules with an apparent molecular weight of 1000 daltons or more The first fraction is divided into a second fraction consisting of molecules having an apparent molecular weight of less than 1000 Daltons. Separate from the fraction. The general cancer-related 80M factor of the present invention is the second component of the ultra-P fluid. Found in fractions.

The terms "apparent molecular weight" and "nominal molecular weight cutoff" mean that ultrafiltration It is a somewhat imprecise method for separating molecules according to their molecular weight within a range of quantities. , positive removed by a filter with a nominal molecular weight cutoff of 1000 daltons. The exact molecular weight depends somewhat on the conformation of the molecule, so it is not used in this specification. Ru. Molecules larger than 1000 daltons in actual molecular weight are 1 e.g. Filters with a nominal molecular weight cutoff of 1000 daltons for relatively long and narrow cases can pass through.

The separation of the second fraction from the first fraction is performed at a nominal molecular weight cutoff of 1000 Daltons. Preferably, this is done by filtering the body fluid through an ultrafilter.

The purity of the preparation of the factor at the ultraf liquid stage or beyond depends on its specific activity. can be described. As used herein, the term "specific activity" refers to a specific fraction of 8 Intracellular fluorescence when used to stimulate lymphocytes that respond to SOM in the 0M test The reciprocal of the amount of protein required to reduce the polarization value to a certain degree, e.g. 20%. is defined as The purpose of purifying factor 80M is to increase the specific activity of factor 80 to more than the specific activity found in crude P solution. The goal is to increase the specific activity of factor M. Therefore, the purification process A step of determining specific activity follows.

The concentration of protein in the examples reported herein is preferably at 220 nm. is determined only approximately by the ultraviolet absorbance at the length, and the factor line Since the dose-response curve is not finalized, the 80% concentration described here in terms of specific activity The characterization of the various purification steps for Factor M is only approximate. However However, protein concentration decreases significantly as the factor moves through various purification steps. Ruga.

activity is relatively unaffected, thereby leading to an increase in the specific activity of factor 80M. It is obvious. Despite the nominal molecular weight cutoff of 1ooo daltons, Ultrafiltration through membranes removes all proteins and large peptides from biological fluids. As long as it removes the vast majority of specific biologically active molecules, the f-fluid is essentially can be described as consisting essentially of pure common cancer-associated SOM-recognition factors. Wear.

The next step in the purification of common cancer-associated factor 80M is a desalting step and an ultraviolet The second fraction obtained from the filtration is loaded onto a chromatographic column that can separate salts. be done. The material loaded onto the column is then eluted from the column with distilled water. and The fraction that elutes with an elution volume of between about 0.3 and 0.5 times the chromatographic bed volume ( 80M factor). The column used in this step has a range of 0 to about 700 da Gel filtration column with a fractionation range of Luton, such as the product name 5ephadex G -10.

The next step in the purification process is another sieving step that separates again according to size. Desalination The SOM-containing material obtained from the process has a content of approximately 1500 to 30,000 Daltons. Another gel filtration column with a coverage area, such as 5ephadex G-50 is loaded. The material loaded onto the column is then treated with a weak aqueous solution of ammonium salts. eluted at The ammonium salt is amthonium bicarbonate, preferably 50mM bicarbonate. Ammonium acid is preferred. Approximately 0.4 to 0.6 times the total bed volume of the chromatograph The fraction that elutes within the range of elution volumes contains the 80M factor and is collected.

The next step in purification is anion-exchange chromatography, which separates by charge. That's about it. The 80M factor-containing material from the previous gel filtration step was transferred to an anion exchange column ( Preferably, on diethylamine ethyl cellulose (DEAR cellulose) Load f. The material loaded onto the column is then colored with increasing concentrations of ammonium salts. eluted from the system. The ammonium salt is preferably ammonium bicarbonate; The increasing concentration of monium salt is 10 mM to 1.0 M ammonium bicarbonate. about The fraction eluting from the column with 0.28-0.31 M ammonium bicarbonate is 80 Contains M factors and is collected.

The final stage of purification is reverse phase high pressure liquid chromatography (FLP-HPLO), It separates by charge and/or hydrophobicity. Typically, DEAE-Se The 80M factor-containing material from the lurose column eluate has dimensions of 220 m x z. 101 RP-HPLC! Column (product name Aquapore RP-300) is loaded on. Elution was then carried out using a combination of two solvents, starting with 70% aqueous 90 volumes of aqueous trifluoroacetic acid (TFA) and 0.1 volumes of water in setonitrile ．． A solution containing acetonitrile with a volume of 0.09 volumes of TFAIO A high concentration gradient is applied. The 80M factor from all starting materials is 70% aqueous. 26 volumes and 0.09 volumes of aqueous TFA in 0.1 volumes in setonitrile. Eluting as a homogeneous peak in the solvent composition of 74 volumes of aqueous TFA, this Apart from this, FLP-HPLO is Peckman's commercial name Ultrasphere OD. It can be carried out on a reverse phase high pressure liquid chromatography column. this color In the case of aqueous 70% acetonate, elution was performed with a few specific solvent combinations, starting with aqueous 70% acetonyl 70 volumes of trifluoroacetic acid and 0.1 volumes of aqueous trifluoroacetic acid in Trill 30 volumes of trifluoroacetic acid followed by a high concentration of the acetonitrile-containing solvent. Performed by concentration gradient. 80M factor when using this column and this solution system Always 43.7 forms of 0.1% aqueous trifluoroacetic acid in aqueous 70% acetonitrile The solvent composition of 56.3 volumes of volume and 0.1 aqueous trifluoroacetic acid Elutes as one peak.

Use of 80M factor The activity of Factor 80M can be determined according to the general test method described in the primary publication. This is confirmed by its effect on lymphocytes potentially reactive to SCM: L, 0erck and B, 0ercek, “App cation” of the Phenomenon of Changes in the 5 truncatedness of Oyloplastmic (SOM) in the Diagnosis of Malignant Disorders :a Review, ”Europ, J, Cancer13, 903-91 5 (1977). The general cancer-related SOM-recognition factor of the present invention can be applied to cancer patient donors. - potentially reactive lymphocytes from KSC! as described in the above publication. When used to stimulate the lymphocytes in the M test, the intracellular fluorescence level of the lymphocytes increases. significantly lower.

The degree of decrease in the intracellular fluorescence polarization value of the stimulated lymphocytes is determined by the degree of decrease in the intracellular fluorescence polarization value of the stimulated lymphocytes. substantially at least 20% even when using If fractions are used, the amount is 50 or more.

Two established methods are important for the proper implementation of the 80M test reported here. It is. These methods involve the isolation of lymphocytes potentially reactive to SCM and the use of fluorophores. The value is to measure itself and convert them into a meaningful number for the 80M test. . These methods are summarized in the following discussion.

(1) Isolation of lymphocytes that potentially react to SCM and potentially react to SOM Isolation of lymphocytes is also performed in a prior patent appl. cation by B, 0ercek & L, 0ercek, 5eri al No, 838,264.　　　March　10゜1986 , entitled"Automated Co11ection Buoy antDensity 5 specifio Ce1Is from Densi ty Gradjents''〇The fraction of these lymphocytes from the general lymphocyte population However, only a relatively small proportion of lymphocytes (approximately 20-25%) respond to the 80M test. Therefore, it is important for proper implementation of the 80M test. Therefore, for undifferentiated lymphocytes The test is to ensure that the lymphocytes that can actually react in the 80M test have completely reacted. Even when I responded.

The decrease in the intracellular polarization value will be observed to be extremely small.

Peripheral blood samples from donors to isolate lymphocytes potentially reactive to SCM Collect in a heparinized tube. After collection, the peripheral blood is treated with iron powder or Treated with carbonyl iron powder, the tube containing the blood and iron powder mixture is placed on top of a magnet. to separate phagocytes along with iron powder from the blood sample. Then a portion of the blood sample is Gradient solution (trade name: Ficoll 1-Trios) ) and centrifugation to remove potentially SCM-reactive lymph based on density differences. Separate the spheres. This separation method has a density of 1.081 t/cd and 0.32 t/cd at 25°C. Centrifugation using a density gradient solution with an osmotic pressure of 00sr+yt1 at 25°C 550X# for 20 minutes at a temperature of . Lymph potentially responsive to SOM Remove the sphere separated cell layer onto the density gradient material using a Pasteur pipette. be recovered by doing so. This material has a variety of heavier Avoid removing density gradient material as much as possible as it contains plasma and cell fractions. Must be careful. Removal of lighter plasma material also reacts with contaminants and SOM. The introduction of non-existent cells into the test sample should be avoided as much as possible to exclude.

Lymphocytes potentially reactive to 8CM are then isolated.

2.3 washing step, initially in 0.9% chloride solution without preservative) and then washed in Dulbecco's complete phosphate buffered saline (PBS). hand.

Hold at 37°C for subsequent use in the 80M test method.

A method for measuring the fluorescence polarization value of a sphere is described in the following publication by B, Oercek & L, Cercek, 5erial No, 867.079゜filed May 27.1986.　entitled　Method　formas uring Pa1arized Fluorescence Emissio ns”.

As described in these documents, S Lymphocytes reacting to CM were placed in a sterile glass tube at 37°C with a known concentration of mitogen, For example, phytohemagglutinin or the common cancer-related SCMs that are the subject of the present invention. Temperature with cancer-related antigens such as recognition factors. Concanavalin A and A Phytohemagglutinin (P) IA as a rhizome mitogen of P. americana ) have been used, but PHA is preferred for the 80M test. This temperature is mixed with mitogens appropriately diluted in a cell suspension of 1-6 x 10 cells/a. or by adding 0.1 d of antigen.

The device runs for 30-60 minutes.

The warmed lymphocytes can then be intracellularly hydrolyzed to fluorogenic compounds in suspension. Suitable non-fluorescent compounds (hereinafter referred to as fluorescein precursors) 1 such as fluorescein/ - diacetate) (FDA).

The fluorescein diaceate has a pH of 7.4 and an osmolality of 0.330 os Used at a final concentration of 0.25mM in complete PBS and acetone or ice. Diluted from a concentrated stock solution prepared in acetic acid.

FD a 0.21117 aliquot of control or stimulated lymphocyte suspension with a syringe. Slowly pour into the beaker containing A substrate solution 31.

Expose cells to FDA for sufficient time (approximately 5 minutes).

Infiltrate the PDA substrate solution into the lymphocytes. Within those cells, a non-fluorescent film Luorescein diacetate molecules are converted to fluorescein molecules by enzymatic hydrolysis. be transformed.

Fluorescent-containing lymphocytes are characterized by the polarizing effect of emitted fluorescent light on the polarizing effect of excitation light. does not depend on the direction of the surface light used to excite the lymphocytes, so it is sensitive to polarized light. Those reactions that occur are isotropic. However, the standard used to measure Current fluorescence polarization measurement devices use vertically polarized excitation light to excite lymphocytes. Because it uses excitation, the measurement method is described in terms of vertically polarized excitation light. It is listed.

When exposed to excitation energy in the form of vertically polarized light, fluorescein The molecules emit fluorescence. Vertically polarized and horizontally polarized emissions measure the relationship between This is the intensity of polarized fluorescence in the vertical and horizontal planes. This is done by measuring the polarization value (P value) according to the linear relationship. Can: However, engineering and XH are on the vertical and horizontal planes, respectively. is the intensity of polarized fluorescence at G passes through the optical system of the specific device used. is a correction factor for unequal transmission of horizontal and vertical components of polarized light. The value of G is , water with the same wavelength as that used for measuring the intensity of horizontally polarized light at 80M. 10 M of fluorescein in PBS excited with squarely polarized light It is determined by dividing by the vertical intensity emitted from the solution. report here The measured G value is 0.42.

stimulated lymphocytes, i.e. the general SOM-associated cancer recognition factors of the present invention. The P value for the lymphocytes determined was that of a control suspension of unstimulated lymphocytes from the same donor. It is compared with the P value. and the P value of stimulated lymphocytes compared to that of control lymphocytes. The percentage decrease in P value is an indication of the SCM-response to the cancer antigen.

Although SOM reactions can be observed over a range of excitation and emission wavelengths, fluorescence When using FDA as the generator precursor, an excitation wavelength of 470 nm and 510 nm It is highly desirable to use an emission wavelength of nm. However, good results It was also possible to obtain this using an excitation wavelength of 442 nm and an emission wavelength of 527 nm.

The spectrophotometer used to measure SOM fluorescence must be highly sensitive and stable. , and the intensity of the polarized fluorescence emission was recorded as a function of time at 80 M The offshore (bulk) concentration of fluorescein in measurements is on the order of IOM ~ 10 M. Therefore, it is necessary to be able to compensate for fluctuations in the intensity of the excitation light. Also, broadband The filter device is difficult to measure in SCM because the SOM response is detected only within a narrow wavelength range. Not suitable for regular use. Set the excitation monochromator to 470 nm and the emission monochromator to 470 nm. When setting the excitation monochromator to 510 nm, the maximum optical threshold of the excitation monochromator is The slit width was 20 nm, and the maximum optical slit width of the luminescence monochromator was 1. It is necessary to set it to 0 nm.

Spectrophotometers are thermally stable because the polarized fluorescent light emission is extremely temperature dependent. A controlled cuvette holder should also be provided. Furthermore, the spectrophotometer uses fluorescent light. It is also necessary to provide means for measuring the horizontal and vertical polarization components of the emitted light.

In the examples presented hereafter, we equipped a thermostably controlled cuvette holder. A Perkin-Nilmer MPF-4 spectrophotometer was used. All measurements were taken at 27℃ I did it. The light source was a xenon lamp.

In the measurement of fluorescence polarization, the emission parallel and perpendicular to the vertical excitation light beam The intensity is alternately increased for approximately 6 minutes with an automatic side photon converter, or to a vertically excited light beam. The vertical emission intensity is recorded at 1, which is 80-90% of the total scale deflection of the recorder. .

Fluorescein leakage from cells and fluorescein packaging in substrate solutions. These readings need to be corrected for background noise. Make this correction For this purpose, the cells were filtered using a 0.22 μm nitrate filter attached to a suitable filter head. The solution is filtered over a cellulose filter. Same fluorescence polarization measuring device Fluorescence parallel and perpendicular to the excitation light is obtained using the Then cell correction full The olecein intensity is obtained from the F solution from the total fluorescence intensity extrapolated to the half time of the F period. It can be obtained by subtracting the values. This extrapolation shows that the amount of fluorescein from cells Due to leakage and natural hydrolysis of FDA, the packground becomes high during temperature. It is necessary.

Separately, 0erceks tapered patent application (Serial No. 867. 079. filed May 27.1986.　entitled “Met” hod for Measuring Po1arized Fluoresc enceEmissions'') A method can be used. In summary.

This method measures horizontally and vertically polarized fluorescence at more than 51 wavelengths. The need to evaluate each example by calculating the intracellular fluorescence from them I've lost it.

Lymphocyte suspension 1.0 at 6X10 cells/N according to the above protocol. m and mitogens/or antigens 0, 1 rLl, FDA as fluorogen precursor. use.

The 80M test was performed using an excitation wavelength of 470nm and an emission wavelength of 510nm. Here, the "standard 80M test" is a common cancer-related 50M factor (its purification method is described here). ) is used as an effective stimulant in the 80M test for the detection of cancer . Therefore, cells from cancer-affected non-fl donors were stimulated in the 80M test. showed significant changes in SCM responses when stimulated by cancer antigens used as agents. Not though.

Cancer-affected donor cells exhibit significant changes in SCM response. This change is When cancer-affected donor cells are stimulated with cancer-associated antigens, the P value decreases and It can be seen as As explained, factor 80M does not detect specific cancer types. (in the 80M test, the f1*!j lymphocytes stimulated with the 80M factor.

Factor 80M does not need to come from a donor with the same type of cancer as the donor of the body fluid it comes from.

When used as a stimulant in the 80M test, SC! The M factor has a P value of at least 1 0chi, in some cases as much as 44.6%. Ps-stimulated lymphocyte activation Recoat P value (F) indicates that Pc was not stimulated by factor 80M for lymphocytes. If Hoot's P value is taken as This indicates the presence of a malignant tumor in the body of the donor of the collected lymphocytes.

The preferred method of using factor 80M as a stimulant in the 80M test is to use Ps and Fluorescence polarization of another aliquot of lymphocytes contacted with mitogen (effect) It consists of determining the SOM reaction ratio, RR8oM, by comparing it with the value PM. here in ”　R80M　=　Ps　72M An FLR of approximately 0.9 or higher indicates that the lymphocyte donor has malignant tumor CM. indicates that it exists.

2 Use in cancer management Common Cancer-Related SCM-Recognizing Factors for Killer Lymphocytes In Vivo Since it is not thought that it participates in the defense of cells, it is a natural consequence that the 80M factor Methods for selectively lowering endogenous activity are easily influenced by killer lymphocytes of cancer cells. can be useful in cancer management by increasing the “What is in-vivo activity? The ability of scM-recognition factors to reduce the cytotoxicity of killer lymphocytes to malignant tumors Taste.

As a first step in such a method, once the lymphocytes of a known or suspected cancer patient are There was a positive reaction with a common cancer-related SOM-recognition factor as a stimulant in the M test. If indicated, a sample of body fluid is taken from the patient and a nominal dose of 1000 Daltons is Pass through a filter with a molecular weight cutoff, collect the fraction passing through the filter, and The fraction was used to stimulate lymphocytes from the same patient in the M study Confirm the presence of SOM-recognition factor. In particular, other clinical indicators indicate the presence of cancer In some cases, it is not necessary to always carry out this test.

Once the presence of factor 80M in the body fluids of cancer patients is demonstrated or inferred, the body fluids By selectively inactivating the factor by reducing the in vivo action of the factor. that body fluid can then be returned to the patient to treat the patient's cancer. can increase resistance to Common Cancer-Related 80M Factors Affect Patients The natural conclusion is that the response in the 80M test will be affected regardless of the type of cancer treated. The in-vivo effects of 0M factors are not limited to the specific type of cancer initially diagnosed, but also to cancers that develop later. It can also increase a patient's resistance to other types of cancer at risk. child This occurs when a patient is treated with immunosuppressive drugs or when a condition such as AIDS occurs. This is of interest when treating patients with already weakened immune systems.

When the body fluid is peripheral blood, one way to reduce the in vivo activity of factor 80M is to The solution is dialyzed to remove peptides with an apparent molecular weight of less than 1000 Daltons. (Factor II: Use an ultrafilter with a nominal molecular weight cutoff of c1000 daltons. This is a method of physically removing it (because it can pass through).

Another way to reduce the in vivo activity of factor 80M in body fluids is to reduce the in vivo activity of factor 80M in body fluids. This is a method of neutralizing or inactivating the virus using specific antibodies. The 80M factor is a polylithic covalently attached to larger carrier molecules such as antibodies and antibodies prepared into complexes. be able to.

Once such antibodies have been cleaved, antibody-forming cells can be isolated and purified using conventional methods. Monoclonal antibodies are produced by cell fusion with a bullidoma. Polykrona Once a monoclonal or monoclonal antibody has been formed, it can be used to neutralize factor 80M. can be used.

Separately, once the antibodies are formed, they are treated with the protein hydrolase papaya can be cleaved into monovalent Fab fragments with a single molecule. Such cutting results in Each fragment molecule created has a site that binds two molecules of different factors. In contrast to virgin antibodies, which are capable of binding only one molecule of the factor. big reason If the formation of antibody-antibody complexes is undesirable, the use of fragment ρ may depend on the application. Therefore, it is preferable to use virgin antibodies. Large antigen-antibody complexes in peripheral blood The presence of coalescence can lead to serum illnesses and other allergic reactions.

3,8 Further uses of antibodies against CM factors Antibodies generated in the management of cancer Yet another method for using cancer in SCM involves targeting anticancer agents to the cancer site. In order to increase the effective concentration of anticancer substances at the site, antibodies with substances with anticancer activity are used. It is to add to. This method uses anti-cancer substances that cause side effects when administered in large doses. It is particularly advantageous when

Anti--8°C antibodies can be prepared by radioimmunoassay, fluorescence immunoassay or enzyme-linked immunoabsorption. Methods such as the probate method.

or precipitation assays such as nephelometry (assuming that the 80M factor is not a monovalent antigen). It can also be used to determine the level of factor 80M in peripheral blood or other body fluids depending on the Ru. Assuming that factor 80M physically associates with cancer cells in some way, Antibodies are labeled with imaging agents such as photodyes or radioactive isotopes.

It can also be used for cancer cell imaging so that cancer cells can be detected in biopsies. Ru. In addition, fluorescently labeled antibodies are used for automatic detection of cancer cells by flow cytometry. can.

Example The following example is Factor 80M, its isolation and purification.

and how to use it. However, the first example is for illustrative purposes only. Please understand that this is not a limitation.

made Active cancer, inverted breast, lung, colon, ovary, cervix, uterus.

Patients with a positive diagnosis of cancer of the larynx or skin (basal cell carcinoma and malignant melanoma) A blood sample of a person was collected in a glass bottle containing beparin (e.g., the product name Vacutai). (nner). Approximately 1 zooxp each of 2011 J of Soera's blood samples. Centrifugation was performed for approximately 40 minutes. Collect the plasma above the sedimented blood cells and collect 10,000 dal. A porous membrane filter with a molecular weight cut-off of 1000 m (e.g.

Pressure was applied to the product (product name Am1co U M2 or YM2 filter) and filtered. . These P solutions were lyophilized or stored at 4°C until further purification.

Example 2: Test of initial purified SCM factor Aliquot of ultra-F solution from each sample of Example 1 The coat contains potentially SCM-reactive lymphocytes obtained from the same donor and the 8 They were set up with lymphocytes whose SCM responses were tested according to the 0M test. Izu In both cases, ultraf fluid reacts characteristically with lymphocytes that react with SOM. Reduced P value (see Table 2) Table 2 80M activity of ultraf liquid of Example 1 Malignant melanoma Malignant melanoma 757 Malignant melanoma Basal cell carcinoma SZO Throat Head Laryngeal Cancer 62-9 Breast Cancer Breast Cancer 76.0 The data in Table 2 are rough limits. Even when present in external fluid, factor 80M is present in lymph nodes that respond to SCM from cancer patients. The decrease in the P value of lymphocytes can be detected by using crude extracts of cancerous tissue or the tissue itself. was made to be at least equal to the decrease in P value seen when stimulated with

The decrease in P value upon stimulation with ultraf-fluid is at least 10 times (that is, the SOM-positive reaction ), which is a characteristic of response. However, the 80M factor is nominally 500 Daltons. Pass through a filter that cuts off molecular weight (product name Am1con UMO5). I didn't pass. These data demonstrate that the activity varies from small molecules such as a single amino acid to large molecules. It demonstrates the small size of the factor while showing that it is large.

Sterile preservative-free water 2 for injecting the lyophilized powder from the sample of Example 1 It was dissolved in d. At this stage, the 80M activity of the preparation was confirmed. and the same tie Active samples from donors with cancer of each type and site were pooled. pooled sample is the product name 5ephadex G-10 which has a fractionation range of 0 to 700 Daltons. Desalted on a 0.9x18 crn column. Samples for each column chromatographic experiment The sample volume did not exceed 25 inches of column volume. Elution is 8-9 crI with double distilled water. A linear elution rate of V'hr was performed. Desalting was performed at room temperature (21-23°C).

Collect 1d fractions eluting between 0.3 and 0.5 times the total volume of the chromatographic bed. The optical density of one fraction was determined. 80M activity was contained within the first eluting peak.

The presence of 80M activity in the peak was confirmed by the 80M test. breast cancer An aliquot of the first eluting peak prepared from the ultra-P solution initially obtained from the patient's plasma. The P value of lymphocytes from breast cancer patients was 86.3 points, which was the drug value in the 80M study. decreased to This indicates the presence of 80M activity.

The dough fractions were collected and lyophilized.

The eluent was further used as a gel fester ram (trade name: 5ephadex G-50). This has a fractionation range of 1500 to 30,000 Daltons). I further refined it. Lyophilized and desalted samples were added to 50mM NH4HCOs. Dissolve and transfer column volume onto a 0.9x18crn 5ephadexG-50 column. The load should be less than 5% of the product.

The linear elution rate was 3 cm/hr. Elution was performed at room temperature. and Eleven fractions were collected that eluted from the column at 0.4 to 0.6 times the chromatographic bed volume. collected. These fractions were tested for 80M activity. The stiffness test results are in Example 4. show. SCM-activity fractionation.

The first determined by the optical density of the fraction 1- after testing the fraction in the 80M test. was included in the eluted peak.

Once the fractions were tested for 80M activity, active fractions from the same cancer were pooled. and lyophilized.

For further purification, the lyophilized sample was added to 10 mM NH4HCOs. It is dissolved to produce fine particles of DE-52 (trade name: Whatman). AE-Cellulose 0.8x26 crn column loaded to less than 4% of column volume. It was. The column contained 10mM aqueous N)14HC0x1Qiu, 10mM From IM N H4) (washed by increasing the concentration to 0108 cm/min. 1d fractions were collected and the optical absorption at a wavelength of 220 nm was determined for each fraction. Ta. Based on its optical absorption, 4.5 to 4.7 times the total chromatographic bed volume The active fractions eluting from the rum were pooled and lyophilized for testing and further purification. It was dry.

Table 3 shows that the 5ephads of Example 3 were initially obtained from donors with various types of cancer. An aliquot of the ex G-50 fraction was used in the 80M test to The results are shown when stimulating the nno-no-ball. Lymphocytes that potentially respond to SCM are G-50 images show the same characteristic responses as they did to previously characterized cancer-related antigens. You can understand what is shown in minutes. This desalted, partially purified protein-containing material is Compared to the crude ultraf liquids whose results are shown in Table 2, they generally exhibit a higher SCM response.

This high SOM response is indicated by a decreased P value.

Table 3 Example 3 5ephadex ()-50 fractions of SCM active lung cancer Breast Cancer 73.0 Lung Cancer Cervical Cancer 69.6 Lung cancer Bronchial diameter 73.6 Laryngeal cancer Tracheal cancer 77.6 Breast cancer Tracheal cancer 80.2 Colon cancer Breast cancer 63.5 Laryngeal cancer Breast cancer 6 3.0 Malignant melanoma Malignant melanoma 74.9 Healthy donor Malignant melanoma 99.3 Healthy donor Colon Intestinal cancer 98.0 Colitis Colitis @! i98.9 g4 Table U, various types of malignant tumors after purification via DEAE-cellulose using factor 80M obtained from a donor with various types of malignant tumors or In the 80M study, it was isolated from a malignant tumor-free donor and stimulated the 90-year-old gland. The results are shown below. As expected, one non-no from a cancer patient (the ball is DEAE- There is a significant decrease in P values in response to scN+ factors purified from cellulose columns. However, lymphocytes from donors without malignancy did not show such a decrease in P values. .

, Table 4 SCM activity of DEAE-cellulose fraction of Example 3 Cancer Milk Cancer 6L2 Breast Cancer Bronchitis 69.5 Breast Cancer Cervical Cancer 69.0 Basal cell skin cancer Basal cell skin cancer SZO healthy donor Conclusion Intestinal cancer 9 8.6 Biliary cystitis Malignant melanoma 100.0 Urine Canal inflammation Neck Cancer 99.8 Insects Tumor Inflammation Intestinal Cancer 980 Benign Breast Long Breast Cancer 97.0 Benign Pituitary Adenoma Brain Cancer Cancer 1’ 80.0 DE-52 General Identification of Example 4 The relevant SCM-active fractions were then reconstituted and Z 1 m x 22 crnHPLc By reverse phase high pressure liquid chromatography (RP-HPLO) using a column It was purified to homogeneity. The column is a product name Aquapore RP-300 (7mm). filled with Chron). The mobile phase used for RP-HPLC purification was as follows: there were: Phase A: 0.1 volume aqueous trifluoroacetic acid (TFA).

Phase B: 0.09 vol. aqueous TFA in aqueous 70 thiacetonitrile.

The lyophilized SCM-active fraction of DB-52 was washed with sterile water (containing preservatives) for injection. First, a 250-μt aliquot was reconstituted with ) and injected into the RP-HPLO column. . The mobile phase flow rate was 50 μt/min and its composition profile was 9 0 volume chi, 10 minutes of phase B's 10 volume chi, then phase B linearly at a rate of 3 volume chi/min. The increase was 30 minutes. Light detected by optical absorption at a wavelength of 220 nm The chemical density peak was detected through a ``nanobore J'' Teflon tube. manually into a 1.5 conical plastic Eppendorf centrifuge tube. collected. The solvent was then evaporated in a vacuum centrifuge. In all cases, the general Cancer-associated SOM-recognition factors were eluted from the column in 74 volumes of phase B.

Example 6: Another ap-HpLc purification of Factor 80M. Product name Ultrasphere ODS sold by Beckman Instruments (5 microns) i’v L fC4,6wa x 25 Crn HP HPLO using LO column and DEAE-528OM-active fraction from Example 4. It can be purified by The mobile phase used with this column was The following was followed: Phase A: 0.1 volume of aqueous trifluoroacetic acid (TFA).

Phase B: 0.1 vol% TFA in aqueous 70% acetonitrile.

Aquapore The mobile phase flow rate for the column is x, oom //min and that its compositional profile was 70% by volume of phase A and 70% by volume of phase B. 5 min of 30 vol. CH followed by 20 min of linear increase in phase B at a flow rate of 3.5 vol.%/min. The same general procedure was followed for this column, except that The optical density peak is Detected at 220 nm and collected manually in a silicided glass test tube, the solvent was Evaporated in a centrifuge. When using this HPLC system, in all cases the cervical One of 19 types of cancer, including squamous cell carcinoma, breast carcinoma, bronchial carcinoma, and malignant melanoma. Purification of common cancer-related SOM recognition factors always yields a single optical density peak of activity.

Eluted with 56.3 volumes of phase B.

In Example 6, the method was carried out by FLP-HPLO on an Ultrasphere column. For 80M activity testing of isolated peptides, peptides were preservative-free for injection. Reconstituted in sterile water without using. After manufacture with these samples.

The 80M activity of SOM-reactive lymphocytes is shown in Table 5.

This fractionation showed the greatest decrease in polarization efficacy values when used to stimulate lymphocytes from cancer patients. Indicates low Two of the three preparations of factor 80M had significantly lower values than the other fractions. It showed a decrease in the polarization effect value by more than 1. As expected, the purified factor is It was nonspecific regarding the type of cancer afflicting the lymphocyte donor.

The purified factor also showed a response when used to stimulate lymphocytes from healthy donors. did not show.

Example 60 SCM activity of RP-HPLO fraction Milk Cancer Breast Cancer 58.9 Breasts Cancer Lung Cancer 57.3 Colon cancer Breast cancer 55.4 Breast cancer Bronchial cancer 68.0 Healthy donor Breast cancer 99.8 Healthy donor Lungs @ 101.0 Example 8: SCM-Activated Trypsin from Factor 80M 16 amino acid long trytic fragment Example 5 isolated from the seminal ffsCM factor.

This fragment was shown to have the total 80M activity of the larger peptide and The sequence was determined by dynamic Edman decomposition. Trypsy of purified factor 80M The cleavage and purification of active fragments were performed by the following method: To prevent absorption loss of peptides during lyophilization.

80M factor was digested with trypsin in the presence of HPLC eluate. trypsin Digestion was performed using 10 wt% trypsin at 0.1 M for 24 h at 37°C. Performed in Tris-HCt buffer (pH=8.3).

The digestate was diluted 4 times with 0.1 volume of aqueous trifluoroacetic acid and Injected into flow HPLO separation device (Applied Biosystems) did. Tryptic fragments were collected using an Aquabore RP-300 column (2000x 2. Separation was performed using IN. For elution of those fragments, The mobile phase solvent was as follows: Phase A: 0.1 vol. aqueous trifluoroacetic acid (TFA) Phase B: 70% aqueous aqueous 0.09 vol. TFA in setonitrile, the mobile phase flow rate was 50 μt/min, and Its composition profile is 96/10 volume Ti of phase A, 4 volume Ti of phase B, followed by A linear elution gradient consisting of a 30 min linear increase in phase B at a rate of 3 vol/min. Ta.

SCM-active tryptic peptide fragments are added to 69.6 volumes of phase B and The sample was eluted with a total volume of 30.4 μt (total volume approximately 30 μt).

Table 6 lists the various essences from Examples 1, 3 and 6 as stimulants in the 80M test. By using preparations of common cancer-associated SOM-recognition factors at the manufacturing stage. The results obtained are summarized below. Lymphocytes from donors suffering from many different malignancies When used with the inventive factor in the 80M test, there was a significant reaction in all cases. There was a response. This reaction is shown in Table 6 for the same lymphocytes that were not treated with the factors. It is shown as the contrast polarization value obtained by performing the sc bi measurement. vinegar. The smaller the value, the greater the response to the factor in the 80M test. . Even with the coarsest preparations of the factors tested, the Ultra-F solution ranged from 18.0 to 37. lchi bias The most highly purified fraction purified by FLP-HPLO showed a decrease in light value and was 44.6%. It showed a large decrease in polarization value. The factors of this invention are unique.

It only results in a decrease in polarization values when used to stimulate lymphocytes in cancer patients.

R,P-HPLC-purified fractions also showed bias when used to stimulate lymphocytes from healthy donors. did not result in a decrease in light value.

Table 6 Example of 80M activity at various sugar fruit stages (Example 9) Sephadex G-5 0 fraction　　　　　19.8-37.0DE-5,27 and loin fraction 18.0-31.0RP-HPLO fraction 310- 44.6 Lymphocytes from healthy donors or non-malignant diseased donors are tested. None of the fractions showed any activity.

The tryptic peptide, the purification of which is described in Example 8, was tested in the standard 80M test. Fully active. This fragment of approximately 5×10 fP (i.e. 5×10? , or 16. A fragment of the OO0 molecule) showed full activity in the test.

When the fragment was isolated from a lung cancer patient, it was found to be small in the 80M test. activity was demonstrated when tested on lymphocytes of lung cancer patients, and as shown in Figure 7. Completely cross-reacted with lymphocytes from adenocarcinoma patients. However, healthy donor lymph Trypsin fragmentation in Example 8 of lung cancer where no reaction was observed when spheres were used. 80M active small cell lung cancer 70.0: 68.0 milk Adenocarcinoma 67.5: 68.0 Healthy donor 101 0: 104.0 The following example demonstrates the stimulation of lymphocytes obtained from patients with different types of cancer. Figure 2 shows the response capacity of the 80M factor in the 80M test when used vigorously. This intersection To demonstrate the response, 2 ml of cells from each of multiple blood samples from cancer patients were collected. Obtained plasma that did not contain any The blood sample was collected on. Those samples cost 1000 Dal A filter with a nominal molecular weight cutoff of M2) for at least 12 hours and stored under sterile conditions at 4°C.

potential from heparinized blood samples from cancer patients, healthy donors, and patients with non-malignant tumors. We isolated lymphocytes that reacted with SOM. Common Cancers Containing Ultra F Fluid-Related To test the activity and cancer specificity of SCM-recognition factors, potential VC8CMs were 0.75j of reactive lymphocytes (5xlo cells/MlinPBS)! /7 Ricoh The sample was incubated with the ultra-P solution at 37° C. for 40 minutes. 0.07517 limit The external f solution was used for each assay method. Measurements of SCM have already been described in the following publications: I went like this: L, 0ercek and B, 0ercek.

“Application of the Phenomenon of Ci Hanges 1nthe 5structuredness of Cytop lasmic Matrjx (SCM) in the Diagnosis f Malignant Disorders: aReview,”Eu rop, J, Cancer 13.903-915 (1977) and i n the prior patent application by B.

0ercek and L, Cercek, 5erial No, 867.0 79. filedMay 27.1986. and entitled″M method for Measuring Po1arized Fluores ence Emissions, “Intracellular fluorescence polarization effect in 80M assay A decrease of at least 10% in the value (P value) was considered a positive response to the stimulating ultraf fluid.

As the data in Table 8 shows, potentially SC from patients with eight different cancer types M-reactive lymphocytes are common cancer-associated SOMs from nine different cancer types. Reacts to ultra-P solution containing recognition factors.

In contrast, ultra-F fluids from healthy donors and non-cancerous diseased donors are positive. No sexual SCM reaction was observed. Also, potentially anti-SCM from healthy donors. Both non-malignant and non-malignant lymphocytes showed no extreme P waves in the 80M test. did not react to.

Decoration Modifying the SOM response of lymphocytes that do not contain malignant tumors by temperature with factor 80M European Journal of Cancer arti cle, 5upra, and application no. 838.264 dated March 10, 1986. Potentially responsive to SCM, as described in issue (B, 0ersek) Lymphocytes were isolated from healthy donor blood samples and treated with Dulbecco's complete phosphate buffer. The cells were suspended in buffered saline solution (PBS) at 5×10 cells/jjl. of these cells The aliquots were heated during the following three times (al to (h) = (a) cells from cancer patients). Plasma without: (b) Am1con with a molecular weight cutoff of 1000 daltons Plasma from cancer patients ultrafiltered through UM2 filter for 12 hours = (c Am1con 0M5 filter with a nominal molecular weight cutoff of 1500 daltons Plasma from cancer patients ultrafiltered for 12 hours through (d) desalted or 5epha Common cancer-related 80 purified via dex CT-10 column stage Factor M: (e) purified via Sephdex G-50 column-stage Factors: (fl D E A E-purified via cellulose column process Factors: (gl Factors finally purified via RP-HPLO step: and (h) Am1con 0M2 with a nominal molecular weight cutoff of 10,000 daltons Plasma from a healthy donor ultrafiltered through a filter. These temperatures 2 I went for 5 hours.

The ability of factor 80M to modify the SCM response of lymphocytes from healthy donors has been demonstrated previously. Determine the SCM reaction ratio (FLR80M) of lymphocytes before and after temperature with each of the fractions of It was shown by doing. 80M to decide on RR Thoroughly wash the mounted cells before contacting them with mitogens or 80M factor. Purified. The presence or absence of modification determines the phosphorus content after a short period of contact with phytoagglutinin (PHA). Polarizing effect values of paracytic suspensions and lymphocytes after short contact periods with substrates containing factor 80M. It was determined by the ratio of the polarization effect value of the sphere suspension. According to the 80M test method, 1. An RR of 0 or less indicates that the donor has a malignant tumor of 80M. A positive indication that RR8CM is present, while RR8CM of 1.1 or higher indicates the absence of malignant tumor. Show that.

Table 9 Modulation of SOM responses of lymphocytes from healthy donors by factor 80M (Example 12) ) Cancer plasma without cells 1.3B 0.80S ephadex G-10 (SOM-active) 1.40 0.80 Sephadex G-50 (SCM-active) 1.38 0.73 DEAE-cellulose (SCM-active) 1.39 0.70RP-HP LC (SOM-Activity) 1.40 0.65 Table 9 is reflected in RR Like, SOMCM Effect of temperature with SOM factor-containing fractions on the response of lymphocytes to factors or phytoagglutinin shows the influence of degree. Lymphocytes that were not pre-temperatured or molecules of nominal 1000 daltons Ultraf fluid from a healthy donor filtered through a filter with a volume cut-off and pre- Temperatured lymphocytes or filters with a nominal 500 Dalton molecular weight cutoff. The ultra-F fluid from the donor with cancer and the pre-equipped lymphocytes were purified through As expected, it showed an RR of 1.35 or more. On the other hand, the SOM factor OM The fraction containing RR8C! M value of malignant tumor The characteristic of lymphocytes initially isolated from patients is reduced to 0,65 to O,SOO values. Effects on innate cytotoxicity of lymphocytes potentially responding to SCM on oligomalignant tumor cells To demonstrate the influence of factor 80M, such lymphocytes obtained from healthy donors were used in cancer patients. with plasma containing factor 80M isolated as previously described from a blood sample of a person. Incubate at 37°C for 25 hours. Aliquots of these lymphocytes were also used as controls. I didn't keep it in a greenhouse. moreover.

Lymphocytes potentially reactive to SCM were obtained from donors with cancer and similarly (some An aliquot was greenhoused with plasma containing 80M factor, and another aliquot was kept as a control. It was kept as a greenhouse and not treated).

After incubation, the cytotoxicity of lymphocytes was determined by the publication (M, R.

Potter and M, Moore+”Natural Cytoxi c Reactivity of Human Lymphocyte 5ubp opulations, 'Imy+unology37, 187-194 ( 1979)). According to this published method , 562 human bone marrow cells labeled with Or as target cells for assay. A cell system was used. Lymphocytes that potentially respond to SCM as effector cells used.

The target cell/effector cell ratio was 1/20. The release of Cr is Indicates that the effector cells are toxic to the target cells. Cytotoxicity is as follows It is determined: However, R8 in the above formula is the release rate of Or in the sample, and Ro is the release rate of Or in the control product. The release rate and RT of cr are determined by the cleaning agent (trade name: Triton X-Zoo). The release of 51Cr in the presence of The results are shown in Table 1O. these The results indicate a nominal 100% of potentially SCM-reactive lymphocytes from healthy donors. The temperature of the ultraf liquid passed through a filter with a molecular weight cutoff of 0 daltons The cells show that their cytotoxicity was reduced by more than 90% in vitro. The m position is cancerous. When used with lymphocytes reacting to latent KS CM from individuals, the decrease in cytotoxicity was The percentage was low at 40-90%.

However, the cytotoxicity of lymphocytes from such cancer patients was very low before treatment; The residual level of cytotoxicity remaining after greenhouse treatment with ultra-P fluid is similar to that of healthy donor lymphocytes. Four speeds were made to the value remaining after the greenhouse. Low levels of cytotoxicity present in cancer patient cells (11 In vivo exposure to factors such as the common cancer-associated SCM-recognition factors of the present invention This was consistent with the decrease in cytotoxicity brought about by

Table 10 In a preferred embodiment, the innate lymphocytes for human bone marrow cell lines of 562 Although the invention has been described in detail in connection with the invention, other embodiments are possible. Therefore, the scope of claim The intent and scope of the following is limited to the description of the preferred embodiments set forth herein. isn't it.

international search report international search report PCT/υ5 g9100961

Claims (52)

[Claims] 1. It passes through a filter that cuts off the molecular weight of nominally 1000 daltons and has a nominal molecular weight of 5. Low molecular weight retained by a filter with a molecular weight cutoff of 0.00 Daltons consists essentially of peptides that cause cancer as measured by standard SCM tests. The intracellular fluorescence polarization effect value of lymphocytes that respond to SCM from a given donor is determined. Substantially pure common cancer-related drug characterized by reducing at least 10% Associated SCM-recognition factor. 2. The factor of claim 1 purified to substantially homogeneity. 3. Substantially, Phe-R1-Lys-Pro-Phe-R2-Phe-R3- Met-R4-R6-R6-R7 [However, R1 is from the group consisting of Asn and Gln R2, R3 and R4 are each independently Val, Leu, and Ile. R5 is selected from the group consisting of Asp and Gln, and R6 and R 7 each independently selected from the group consisting of Asn and Gln). A general peptide characterized by consisting only of peptides having 13 amino acid residues. Cancer-related SCM-recognition factors. 4. the factor is a lymphocyte that reacts with SCM obtained from a donor free of malignant tumor; When contacted, lymphocytes react with cancer-associated antigens in the SCM test, but Modify the SCM response of lymphocytes that respond to SCM but do not respond to itodienes. The factor according to claim 3, which has decorative properties. 5. Natural assay of potentially SCM-reactive lymphocytes to the human bone marrow cell line K562 The property of reducing in vitro toxicity by at least 90% as measured by the release of 51Cr. The factor according to claim 3 comprising: 6. (Asx2, Glx3, Ser, His, Gly5, Thr, Arg, Al a3, Tyr, Met, Val3, Phe2, Ile, Leu3, Lys8) 4. The factor according to claim 3, which has an approximate amino acid composition. 7. A 16 amino acid segment within the factor has the amino acid sequence Phe, Asn-L ys-Pro-Phe-Val, Phe-Leu-Met-Ile-Asp-G in-Asn-Phe-Ser-Lys and does not contain the amino terminus of the factor. The factor described in item 3 of the desired range. 8. A 15 amino acid segment within the factor has the amino acid sequence Phe-Asn-L ys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp-G Claims having ln-Asn-Thr-Lys and not including the amino terminus of the factor The factor described in Section 3. 9. 4. The factor according to claim 3, which contains substantially only one peptide. 10. Substantially, Phe-R1-Lys-Pro-Phe-R2-Phe-R3 -Met-R4-R5-R6-R7-R8-Lys [However, R8 is Ser and Th at least 15 amino acid residues containing the sequence selected from the group consisting of The factor according to claim 3, which comprises only a peptide that 11. 11. The factor according to claim 10, which contains substantially only one peptide. 12. Substantially, Phe-Asn-Pro-Phe-Val-Phe-Leu- Peptide containing the sequence Met-Ile-Asp-Gln-Asn-Thr-Lys 11. The factor according to claim 10, which comprises only peptide. 13. 3. The factor according to claim 2, which contains substantially only one peptide. 14. Substantially, Phe-Asn-Lys-Pro-Phe-Val-Phe- Contains the sequence Leu-Met-Ile-Asp-Gln-Asn-Thr-Lys. 14. The factor according to claim 13, which consists only of a peptide having the following. 15. Substantially, Phe-R2-Lys-Pro-Phe-R3-Phe-R3 -Met-R4-R5-R6-R7-R8-Lys [However, R8 is Ser and Th at least 16 amino acid residues containing the sequence selected from the group consisting of The factor according to claim 3, which comprises only a peptide that 16. 16. The factor according to claim 15, which contains substantially only one peptide. 17. Substantially, Phe-Asn-Lys-Pro-Phe-Val-Phe- Contains the sequence Lru-Met-Ile-Asp-Gln-Asn-Thr-Lys. 16. The factor according to claim 15, which consists only of a peptide having the following. 18. 18. The factor according to claim 17, which contains substantially only one peptide. 19. Essentially, the amino acid Phe-Asn-Lys-Pro-Phe-Val- Phe-Leu-Met-Ile-Asp-Gln-Asn-Phe-Ser- The factor according to claim 18, consisting of a substantially pure peptide consisting of Lys. . 20.5 x 10-2 fetograms of peptides suffered from cancer in the SCM test at least 3 when used to stimulate SCM-associated lymphocytes from donors in 20. A peptide according to claim 19, which reduces the polarization effect value by 0%. 21. (a) A suspension of lymphocytes obtained from a mammalian donor as claimed in claim 1 or contacting the common cancer-associated SCM recognition factor of item 3; and (b) Cytoplasmic map of lymphocytes obtained from the step of contacting the suspension of lymphocytes. a step of determining a decrease in the degree of organization of trix. A method of testing whether a donor has a malignant tumor using lymphocytes obtained from the donor . 22. The factor is substantially Phe-R1-Lys-Pro-Phe-R2-Ph e-R3-Met-R4-R5-R6-R7 [However, R1 is from Asn and Gln R2, R3 and R4 are each independently selected from the group consisting of Val, Leu and R5 is selected from the group consisting of Asp and Gln, and R6 and R7 each contain a sequence independently selected from the group consisting of Asn and Gln. Claim 2 consisting only of peptides having at least 13 amino acid residues The method described in Section 1. 23. The factor essentially has the amino acid sequence Phe-Asn-Lys-Pro-Phe -Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Thr - The method according to claim 22, comprising a substantially pure peptide consisting of Lys. Law. 24. The factor essentially consists of the amino acids Phe-Asn-Lys-Pro-Phe- Val-Phe-Leu-Met-Ile-Asp-Gln-Asn-Phe- Claim 22 consisting of a substantially pure peptide consisting of Ser-Lys How to put it on. 25. A 16 amino acid segment within the factor has the amino acid sequence Phe-Asn- Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp- Gln-Asn-Phe-Ser-Lys and does not contain the amino terminus of the factor The method according to claim 21. 26. A 15 amino acid segment within the factor has the amino acid sequence Phe-Asn- Lys-Pro-Phe-Val-Phe-Leu-Met-Ile-Asp- Claims having Gln-Asn-Thr-Lys and not including the amino terminus of the factor The method according to paragraph 21. 27. Determining a decrease in the degree of organization of the cytoplasmic matrix comprises: (a) Measuring the fluorescence polarization effect Ps of an aliquot of lymphocytes that have already been contacted with the factor. (b) determining a second aliquot of lymphocytes that has not been contacted with terminal factors; Measuring fluorescence polarization effect Pc; and (c) A malignant tumor is present in the lymphocyte donor with a Ps/Pc ratio of approximately 0.9 or less. Claim 21, comprising the step of determining a ratio of Ps/Pc indicating that The method described in section. 28. Determining a decrease in the degree of organization of the cytoplasmic matrix comprises: (a) Measuring the fluorescence polarization effect Ps of an aliquot of lymphocytes that has already been contacted with the factor. Step of determining: (b) phytohemaglunitin, concanavalin A and Americana Lymph that has been contacted with a mitogen selected from the group consisting of mitogenes measuring the fluorescence polarizing PM of a second aliquot of spheres; and (c) The lymphocyte donor has a malignant tumor with an RRSCM of approximately 0.9 or less. Determining the reaction ratio RRSCM of SCM as the ratio of PS/PM showing 22. The method according to claim 21. 29. (a) Separating potentially SCM-reactive lymphocytes from a blood sample: (b) Contacting the separated lymphocytes with the factor set forth in claim 1 or 3. The process of stimulating lymphocytes by: (c) The stimulated lymphocytes are treated with a fluorophore precursor and the precursor penetrates into the lymphocytes. for a sufficient time to hydrolyze the fluorogenic compound by intracellular enzymatic hydrolysis. A step of producing stimulated fluorescent-containing lymphocytes by touching: (c) stimulated fluorescence-containing lymphocytes by exciting them with polarized light; Process of generating fluorescent light in the bulb: (e) Vertically and horizontally polarized light from fluorescent lymphocytes. A method for determining the polarizing action value of lymphocytes that emit fluorescent light by measuring the emitted fluorescent light. degree: and (f) Determined polarization effect values of stimulated fluorescence-containing lymphocytes and step (b) ) The same donor underwent steps (a), (c), (d) and (e) except for compared to the polarization effect value of a control aliquot of lymphocytes from A blood sample characterized in that it consists of a step that indicates the presence or absence of cancer in the donor's body. A method of screening blood samples to indicate the presence or absence of malignant tumors in a donor's body. 30. Lymphocytes are excited by vertically polarized light, and the polarization effect value is expressed by the following relational expression: The method according to claim 29, wherein P=(I V-GIH)/(IV+GIH) However, IV and IH in the above equation correspond to the vertical plane and water, respectively. The intensity of the polarized fluorescence in the plane, and G is the polarization passing through the optics of the spectrophotometer. is a correction factor for unequal transmission of horizontal and vertical components of . 31. 30. The method of claim 29, wherein step (b) and step (c) occur simultaneously. 32. (a) Treating body fluids containing SCM-recognition factors to selectively inactivate them. reducing the in vivo action of the factor by: and (b) increasing the patient's resistance to cancer by returning said treated body fluid to the patient; at least one of the body fluids of the patient is a common cancer. - A method of curing cancer patients comprising related SCM-recognition factors. 33. If the body fluid is peripheral blood and the process for processing the body fluid is less than 1000 daltons, Consists of dialysis of peripheral blood to remove peptides with multiplied molecular weight; their removal from the blood The method according to claim 32, wherein the SCM-recognition factor is selectively inactivated by . 34. The step of treating the body fluid involves converting the SCM-recognizing factor in the body fluid into an antibody specific to the factor. 33. The method of claim 32, comprising neutralizing with. 35. The step of treating the body fluid converts the SCM-recognition factor in the body fluid into an SCM-recognition factor. Fab fragments of antibodies specific to SCM-recognition factors capable of monovalent binding 33. The method of claim 32, comprising neutralizing with 36. (a) A general cancer-related SCM-recognition factor according to claim 1 or 3. Labeling an antibody against with an imaging substance: and (b) It is characterized by a step of performing image diagnosis of cancer cells using labeled antibodies. An image diagnosis method for cancer cells. 37. (a) A general cancer-related SCM-recognition factor according to claim 1 or 3. and (b) labeling an antibody against cancer cells to direct the anticancer substance to cancer cells. A labeled antibody is given to a cancer patient so that the labeled antibody can bind to the patient's cancer cells. A method for directing an anticancer substance to cancer cells, the method comprising the step of administering an anticancer substance to cancer cells. 38. General cancer-related SCM of claim 1 or 3 in immunoassay - Using recognition factors to determine levels of common cancer-related SCM-recognition factors in body fluids how to. 39. 39. The method of claim 38, wherein the immunoassay is a radioimmunoassay. 40. 39. The method of claim 38, wherein the immunoassay is a fluorescent immunoassay. 41. 39. The method of claim 38, wherein the immunoassay is an enzyme-linked immunosorbent assay. 42. 39. The method of claim 38, wherein the immunoassay is a precipitation immunoassay. 43. 43. The method of claim 42, wherein the precipitation immunoassay is a turbidimetric assay. 44. 43. The method of claim 42, wherein the precipitation immunoassay is a turbidity assay. 45. (a) Obtaining body fluids of a donor suffering from cancer: (b) 1000 Dalt. A first surface of body fluid consisting of molecules having an apparent molecular weight greater than 1000 da Separating from a second surface consisting of molecules having an apparent molecular weight of less than or equal to Luton: and (c) purifying the second fraction to substantially pure common cancer-associated SCM-recognition factors; Manufacturing a general cancer-related SCM-recognition factor characterized by the step of obtaining how to. 46. Claim 45, wherein the body fluid is selected from the group consisting of peripheral blood, urine, and plasma. How to put it on. 47. The separation of the second plane from the first plane is achieved by a nominal molecular weight cutoff of 1000 Daltons. Claim 45: By filtration of body fluids through a to-off ultrafilter. Method described. 48. In addition, gel filtration has a fractionation range of 0 to 700 Daltons and can separate salts. Load the column with the second fraction, elute the loaded material with distilled water, and chromatograph. Collect the portion that elutes at an elution volume between about 0.3 and about 0.5 of the total bed volume of the feed. desalting the second fraction by Generally in the collected desalted portion for the specific activity in the second fraction of Section 45 46. The method of claim 45, which increases the specific activity of cancer-associated SCM-recognition factors. 49. moreover, (a) collected in a filtration column with a fractionation range of about 1500 to about 30,000 Daltons; Process of loading the collected desalination section: (b). The material loaded onto the column in step (a) is treated with a weak aqueous solution of ammonium salts. Elution from the column: and (c) Elute with an elution volume between about 0.4 and about 0.6 times the total bed volume of the chromatograph. the collected portion of claim 48. Common cancer-related S in eluate collected for specific activity in desalted areas 49. The method according to claim 48, which increases the specific activity of CM-recognition factor. 50. moreover, (a) Transfer the collected eluent to a diethylaminoethyl cellulose anion exchange column. (b) The material loaded onto the column in step (a) is loaded onto the column at a high concentration. Elution from the column with a monium salt: and (c) the portion eluting from the column with about 0.28M to about 0.31M ammonium salt; 49, thereby collecting the collected eluent of claim 49. Common Cancer-Related SCM-Recognition in Eluates Collected for Specific Activity in 50. The method according to claim 49, wherein the specific activity of the cognitive factor is increased. 51. Furthermore, by reverse phase high pressure liquid chromatography, claim 50 Substantially uniform distribution of common cancer-associated SCM-recognizing factors from collected eluates of 51. The method of claim 50, comprising the step of purifying. 52. The method according to claim 45, 48, 49, 50 or 51. Substantially pure generic cancer-associated SCM-recognition factor manufactured by.