DE1903050A1 - Method for the detection and localization of neoplastic cell material - Google Patents

Description

PATENT ADVOCATE DR. HANS-GUNTHER EGGERT, DIPLOMA CHEMIST

5 KDLN-LINDENTHAL PKTBR-KINTGEN-STRASSE 2

20 »January 1969 Eg / Ax

Allan Roy Goldberg, Whiffletree Farm, North Stanwich Road, Greenwich, Connecticut, U, S.A.

Max Marcel Burger, lol Red Hill Road, Princeton, New Jersey, USA

Method for the detection and localization of neoplastic cell material

The invention relates to a method for identifying and differentiating neoplastic cells Cells from normal cells. Neoplastic cells are Cells that are responsible for abnormal neoplasms, these neoplasms are commonly called malignant Neoplasm or malignant tumors are characterized, and the malignant tumors and malignant neoplasms are called Called cancer. The term "cancer" denotes a group of diseases, all of which are the transformation of normal body cells in abnormally growing parasite cells. These abnormal or neoplastic Cells are basically present in all types of cancer and the basis of this identification test,

Cancer ranks second among the causes of death in the United States today. According to the report "President's Commission on Heart Disease, Cancer and Stroke" was cancer in 1963 for 285,362 deaths or 15, ^ of all deaths responsible, and at the At present, cancer will affect one in every four Americans currently alive. These Deaths are caused by cancer, which is numerous

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affects different places in the human body ·. To. the previously known cancers include cancer of the Lungs, prostate, colon and rectum, stomach, pancreas, lymphatic system, whereby it is referred to as a lymphoma, which is a neoplastic Disruption of the lymphatic tissue is *, including plasmocytic lymphoma, follicular lymphoma, the lymphoplastic lymphoma and lymphocytic lymphoma include, Leukemia, cancer of the rectum, bladder, urethra, the oral cavity, throat, breast, uterus, ovaries and the liver and ductile ducts In addition to the designation of the types of cancer near the organs that are attacked by it, the disease can persist labeled according to the specific tissues that are attacked. In particular there is a carcinoma, that is a neoplasm consisting of epithelial cells, which tend to infiltrate the surrounding tissues and form metastases. There is also the acinous Carcinoma, which is a carcinoma with a grape-shaped structure, adenomatosis, a type of cancer, the one forms a sac-like granular structure, alveolar carcinoma in which the neoplastic cells appear in groups, which are enclosed in the connective tissue, chorionic carcinoma, colloid carcinoma, in which the cells undergo colloid degeneration have experienced cylinder cell carcinoma, where the cells are cylindrical, ductal carcinoma in which the cells attack the ducts, the cephaloid carcinoma, which affects a soft, brain-like structure, epibulbar cancer, which starts at the edge of the cornea and spreads over the cornea and conjunctiva of the Eye, epithelial carcinoma, glandular carcinoma, in which the cells become glandular or Secretion-forming type include, mentioular carcinoma, scirrhous carcinoma of the skin, lipomatous carcinoma, a Carcinoma, which is characterized by having a lot

Containing fat, mastidoid carcinoma, the one fast growing type of breast cancer is medullary cancer, Periportal carcinoma, in which the cancer affects the liver infects and extends along the portal vessels and around the portal vessels, the carcinoma of the sons di · the nose and the paranasal sinuses, the sairrhö- ••• carcinoma, which is characterized by a hard structure consisting of connective tissue filled with alveoli, and the · carcinoma tuberosum, a carcinoma of the Skin.

In addition to carcinomas, there are sarcomas, which are tumors in the form of a new formation that resembles embryonic connective tissue. Some of the more common sarcomas are Saroom adipoeus, which contains a copious amount of fat, the alveolar sarcoma, which is a networked fibrous scaffold which includes groups of sarcoma cells, betryosarcoma which affects the vagina, ohloromatous Sarcoma, which affects the periosteum of the skull, the tascial sarcoma, which occurs in the fascia around the bindings stands, the Hodgkinohe sarcoma, the leAocytic sarcoma, i.e., leukemia, lymphatic sarcoma, i.e., lymphosarcoma, osteoblastic sarcoma, which produces tissues that are knot-like or cartilaginous, that osteogenic sarcoma, which includes tumors found in the bone occur and arise from the bone cells, the polymorphic sarcoma that contains a variety of different cells, reticulum cell sarcoma, a malignant one Lymphoma, and spindle cell sarcoma, in which the cells are spindle-shaped.

All of the above types of cancer have one

. common basis, namely the neoplastic cell, which, as in the above mentioned section of the Presidential Commiesion e 'mentioned, undergoes a fundamental biological change that distinguishes it from normal cells.

It was hypothesized that the

BATH ORIGINAL

The change is a permanent change in the nucleic acids contained in the cell. In particular, it is believed that the change affects deoxyribonuoleic acid and ribonucleic acid. Deoxyribonucleic acid is a component of cell genes, and ribonucleic acid has the task of producing enzymes and proteins. There is also a certain change in the morphology of the cell membrane that surrounds all cells ₀ This biochemical change in the structure of the membrane of nedplastic cells applies as responsible for the loss of contact inhibition that is present in neoplastic cells as opposed to normal cells and may be the reason for some differences in the cell division process between neoplastic and normal cells «

Much effort has been made in the past to develop a relatively simple test which, using a rapid laboratory or surgical technique, can detect and distinguish between neoplastic cells and normal cells. These efforts led to the development of tests that included injecting blood into chick embryos. In the chick embryo test, if the blood is from a person who has cancer, the chick embryo will either be killed or a chick will be made with conspicuous deformities blood van comes a healthy patient, the chicken embryo is developing normally. Another test method uses bacteria whose growth is stopped by cancer cells but not by normal cells. Other methods include the Papanicolaou vaginal cytology test, commonly referred to as the Pap test, the X-ray examination with and without the use of opaque materials, the use of a tumor-specific dye, the use of radio-

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active materials, the analysis of blood for enzymes, antigens, antibodies, polysaccharides, glycoproteins, Debris from normal and tumcr ^ pweben, proteins, albumins, Globulins, vitamins, trace metals, amino acids, fats, lipids, cell toxins, etc. «, microscopic examination of tissue and urinalysis. All of these! Tests are geared towards finding some differentiator between normal patients and cancerous patients.

Some of these tests may not have been reliable, but they all have the common disadvantage that they are for a very specific type of tumor, cancer

specific
or MaligniTJät / are. In other words, there has not been a test that a doctor can use to determine in every case the presence of any type of cancer in a living organism.

The invention relates to a method for the detection of neoplastic cells, which makes it possible individual cancer cells in the presence of larger numbers of normal cells through the special compound detect any cancer cells with a labeled agglutinin.

The invention also relates to a glycoprotein agglutinin which enables the detection of neoplastic cells and unlabelled or with a fluorescent material, a visible dye, or a radioactive one Material can be marked. -

The invention also relates to

a method to differentiate between neoplastic cells and normal cells by adding a glycoprotein to these cells;

a test for cancer cells by adding a substance, those specific to the neoplastic cell and not

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reacts with the normal cell;

a method of differentiating between neoplastic Cells and normal cells by the addition of a fluorescent chemical substance produced by the emission of. Fluorescence is detectable when it comes into contact with neoplastic cells;

a method of differentiating between neoplastic cells and normal cells by treating the cells with a radioactive chemical compound that can be detected by emitting radiation when with neoplastic cells come into contact; a method of distinguishing between neoplastic Cells and normal cells by delivering a chemical compound to the cells that produce a stain Consequence visible by an optical method when neoplastic cells are present; a method of differentiating between neoplastic cells and normal cells by treating the cells with a fluorescent G-lycoprotein, which when it comes into contact with neoplastic cells, these Makes cells fluorescent, but not normal cells;

a method to differentiate between neoplastic and normal cell material, this cell material is brought into contact with a G-lycoprotein that Radiation emitted when it comes into contact with neoplastic cellular material;

a method of distinguishing between neoplastic and normal cells, using the cells with a. G-lycoprotein, which, when neoplastic cells are present, forms a color which can be determined by any method that enables the sight and measurement of visible radiation, ZeB, the naked eye, microscopes or spectrophotometers; a method to differentiate between neoplastic cell material and normal cell material, where the Z ₆ Il-

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material is brought into contact with a glycoprotein which agglutinates the neoplastic cell material but not the normal cell material _e

The invention is based on the differentiation of neoplastic Cell material from normal cell material by reaction of a cell material with one for neoplastic

directed
Cell-specific substance / which responds visibly or measurably when neoplastic cells are present. As a substance which is specific for neoplastic cells, a glycoprotein is used for the purposes of the invention, which is isolated from wheat germ or a raw preparation of wheat germ lipase. The term "glycoprotein" used here denotes an agglutinin which is extracted from wheat germ or its derivatives and specifically reacts with neoplastic materials. The glycoprotein is prepared as follows χ The wheat germ or a raw preparation of wheat germ lipase is suspended in a solvent such as water, alcohols, organic solvents, mixtures of these solvents, or any other suitable solvent. The suspension is then ground in order to homogenize the material in it. The suspension is then ^{heated to about 75 °} C. and then centrifuged while removing the precipitate formed. The suspension can be heated to any temperature at which the biological activity of the glycoprotein is not reduced or the glycoprotein is not deactivated. Ammonium sulfate, calcium sulfate, ammonium chloride, ammonium nitrate, calcium chloride or other salts suitable for salting out purposes are then added to the above liquid in such an amount that the glycoprotein is salted out, which in turn results in a precipitate which is centrifuged out. The speed of the centrifuge used is not critically important. Anyone is suitable

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Speed enough to break away. The precipitation from the treatment with the salt is then dissolved in water and then dialyzed against water in the cold by conventional methods, concentrated and redissolved in water, alcohol, organic solvents, their mixtures or other suitable solvent systems and passed through an exchange medium. The temperatures used in this dialysis method are normally between about ⁰ and 4 ° 0, but the dialysis can also be carried out at other suitable temperatures.

Separation media are suitable which enable molecules to be separated on the basis of a difference in their relative size or charge. For example, the gel filtration media with the trade names “Biogel” and “Sephadex” and the resins diethylaminoethyl cellulose and carboxymethyl cellulose are suitable. The usual methods of protein purification are also suitable for purifying the desired glycoprotein, for example electrophoresis on any type of carrier such as starch, foam rubber and polyacrylamide. Chromatographic methods, which are selective Adsorpttion enable the desired compound _e as materials are also suitable, for example, calcium phosphate often used alumina gel, diatomaceous earth, starch and hydroxyapatite in the chromatographic methods ·

In addition to the above purification method, the useful glycoprotein can be prepared by any conventional precipitation purification method using the above purification media in addition to other conventionally sized differentiating materials. The desired glycoprotein can also be obtained by other peeling methods commonly used in conjunction with proteinaceous materials, i. _E. by

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Salt precipitation, isoelectric precipitation and precipitation with organic solvents. Furthermore, the desired Glycoprotein can be obtained in pure form by means of a hapten elution method, i.e. by application a hapten on a support, absorption of the preparation containing the impure. Contains agglutinin, to the hapten bound to the carrier and elution of the agglutinin specifically with a soluble hapten.

The product obtained by this extraction and purification is a pure glycoprotein that has a maximum optical density of 278 ma. And a minimum optical density of 252 nm. Furthermore, no absorption was found above 300 mu (see FIG. 1). The extinction coefficient at 280 µl was 15.0 and the ratio of the optical density at 280 mu to the optical density at 260 mu was 1.34 · Further, it was found that a typical fluorescence excitation maximum occurred at 280 mu and a maximum emission occurred at about 340 mu the G-lycoprotein has a sedimentation coefficient of 2.74 at 20 ⁰ O in water, a diffusion coefficient of 9.2 χ 10 cm / sec. at 20 ⁰ O in water, a partial specific volume (from pycnometry) of 0.71 rnl / g and a partial specific volume (from the amino acid composition) of 0.72 ml / g _e The intrinsic viscosity (intrinsic viscosity) was 8 to 10 ml / cm sec. This Glcoprotein has a molecular weight of about 26000, a friction ratio of 1.2 and an axial ratio of 4-5 · Further, the intrinsic viscosity of the glycoprotein about 9 at 25 ⁰ C. The glycoprotein consists of all the common amino acids (see table) and contains large Amounts of glutamic acid and aspartic acid, namely 15 residues / molecule, respectively. The carbohydrate portion ($ 3-5) of the desired glycoprotein consists almost exclusively of glucose units.

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Table I Amino acid composition of wheat germ agglutinin

amino acid

Calculated number of residues assumed per molecule (molecular number of weight = 26000) residues

Duration of hydrolysis * 24 hours 72 hours

Lysine 7.90 3.92 6.91 8-9 14th Histidine 2.86 2.23 3 Amide NH, 25.99 - 26th Arginine 9.75 9.65 10 25th Asparagine - 20th acid 14.28 14.17 15th 14th Threonine 7.82 7.54 8-9 14th Serine 12.44 11.05 13 - Glutamic acid 19.0 19.0 19th Proline 14.70 14.39 15th 13th Glycine 24.10 24.05 24 - Alanine 19.41 18.94 19 - Half, cystine 13.60 13.20 . 13 - Valine 11.71 12.98 13 - Methionine 3.67 3.81 4th Isoleucine 4.54 4.80 5 Leucine 12.25 12.13 12 - Tyrosine 6.46 6.95 7th Phenylalanine 4.81 4.88 5 Tryptophan ^a 4th

a) Determined by spectrophotometry

* The acid hydrolysis was carried out in an ^{oil bath kept constant at up to 106 °} C. for 24 hours and 72 hours according to the standard method for the Beckman amino acid analyzer for determining acidic and basic amino acids. The cystine half-residues were determined by oxidation with performic acid according to Hirs et al., Chem. JMjJ, 611 (* 956).

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In the method according to the invention, this pure glycoprotein can be combined with a compound that fluoresces, with a compound that emits light in the visible ultraviolet and infrared spectrum, with a radioactive isotope labeled or used as a pure chemical substance in the test according to the invention.

The particular type of radiolabel used in conjunction with the glycoprotein is depending on the way in which the test is to be carried out »In detail, the glycoprotein can be mixed with a light isotope, a radio-opaque substance, or a heavy isotope can be labeled * The choice depends on the specific conditions under which the test is to be carried out. The glycoprotein can with a heavy isotope of elements such as iodine, cerium, xenon, cesium, barium, etc., by mixing the glycoprotein with an alkaline solution, e.g. sodium carbonate, according to the method described in "Proceedings of the Society of Experimental Biology and Medicine "80, 74-1 (1952) is described. A heavy

131 rer isotope as a marking material, such as iodine ^, that

131

duroh addition of iodine to sodium iodide and sodium nitrite solution is prepared is added dropwise with stirring to the glycoprotein solution. The received Product is then poured through an ion exchange column, the drainage of which is made isotonic with sodium chloride will. This isotonic solution is then filtered, leaving a radioactive encoded glycoprotein is obtained.

Further known methods of labeling proteins, e.g. the glycoprotein according to the invention, are Methods described by L. Lutwack in Proceedings of the Society of Experimental Biology and Medicine 80, 741 (1952), by R.C. Gilmore jr. et al in "Nucleonics" 12, No. 2, 65 (1954) and by McParlane in "Biochemical Journal" 62, 135 (1956).

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It is also possible to mark the glycoprotein with a light isotope, e.g. H or C ^ by reacting the glycoprotein with acetic anhydride marked in this way. This can be achieved by adding marked acetic anhydride to an alkaline solution of the glycoprotein and extracting the product by chromatography * The marking can also be carried out by the methods of RoFoColeman et al (Journal of Biological Chemistry 2A, 3652 (1966)) or S ₀ P ₀ Colowick et al ("Methods in Enzymology", 17, page 251, Academic Press, New York 1957).

Another alternative "in radioactive labeling of the glycoprotein is the use of a radio-impermeable substance, e.g. iodine, which is used for labeling of the glycoprotein can be used in a manner similar to the labeling of iodine.

Besides labeling the compounds with a radioactive substance, that used according to the invention can be used Glycoprotein can also be reacted with a fluorescent compound. As a fluorescent agent are for example fluorescein isothiocyanate, 5-dimethylamino-1-naphthalenesulfonyl chloride or Lissamine-rhodamine-ß-200-chlQrid or other similar reactive compounds that fluoresce. These compounds can either be in free form or on "Celite" bound can be used. "Celite" is a silica or diatomaceous earth, also called infusor earth and kieselguhr is called »

The glycoprotein can generally be labeled with the fluorescent compound by reacting an alkaline solution of the glycoprotein with the fluorescent material on kieselguhr. After the reaction has ended, the unreacted material

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rial removed by centrifugation and the labeled Glycoprotein purified by any conventional method. The marking with fluorescent material after this method and other useful methods are described by Rinderknecht in "Ultra-Rapid! Fluorescent Labeling of Proteins ", Nature 193, 167 (1962) and" Experientia ", VI, pp. 4-30 (1960)

The glycoprotein can also be reacted with a dye that is in the visible color range, z »B, with fluorodinitrobenzene, trinitrobenzene, sulfonic acid and diazonium-1,4-tetrazole. In particular, a substance such as fluorodinitrobenzene can interact with the glycoprotein reacted and purified in an alkaline medium, producing a yellow product that Visible to the human eye «Special methods used to mark the glycoprotein with a visible dye are suitable are described by OoLowick et al. at the aforementioned literature site "Methods in Enzymology" and F.Sanger in "Biochemical Journal "39., 507 (1945). That in this way Labeled glycoprotein can be seen with the naked eye or by other conventional optical methods including microscopic and photometric methods are perceived «.

In the method according to the invention either the labeled or the unlabeled glycoprotein is used, which is made to react with a cell material in such a way that normal cell material is replaced by neoplastic Cell material is differentiated. The test method can can be carried out in vitro or in vivo. For example, a pathologist could use one of the glycoprotein substances on a tissue section taken from a patient by biopsy and the sample for signs of fluorescence, radioactivity, or the visible dye depending on the label of the

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observe the glycoproteins »Furthermore, the test could be carried out on a suspension of cell material obtained by surgery, whereby the surgeon would wash the suspect tissue free of surrounding fluids ₀ The glycoprotein could then be added to the tissue while the surgeon determines whether agglutination, fluorescence, radioactivity or the visible dye is occurring depending on the type of label of the glycoprotein used. The test method according to the invention further comprises administering the labeled glycoprotein to a patient who is suspected of having cancer. The specific for neoplastic cells would be targeted substance in the region of a neoplastic formation, and could be observed on the basis of radioactive emissions could _e particular a preparation as described in Example 2, made isotonic with Matriumchloridlösung and through a sterile Seitz Manteufel- Filters are filtered to obtain a sterile isotonic solution for injection. »Approximately 5 ml of this preparation could then be injected into the patient, who would be observed for a possible reaction. The patient's body would then be scanned with a Geiger counter, which is observed at the site of the krebsigeti tissue for a higher level of radioactivity than the rest of the body. This would be an indication that the radioactive glycoprotein has isolated in the area of the neoplastic material because it is specific for neoplastic cells. Some radiation can be observed all over the patient's body, however, this is due to the agglutination of other cellular material containing the tumor-specific substance and an acetylglucosamine

However, due to the large difference in response, it can be assumed that this substance is used for the? test to determine

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neoplastic material is valuable «The test method could also be applied to a suspension of cellular material from a patient suspected of being cancerous has been removed. In this particular case the suspension would be treated with the glycoprotein and up the characteristic reaction observed when neoplastic material is present "

The invention is also feasible that radio-opaque Glycoprotein administered to a patient. The labeled glycoprotein could then, if Cancer is present, detected in an isolated area with the help of a fluoroscope or on the X-ray screen will. In particular, a person suspected of cancer could use a sterile isotonic solution of the according to example 6 prepared glycoproteins are injected. An x-ray can be of the suspect District of the body would be made, and a mass of radio-opaque material would be in the exact location of the Cancer tissue detected. In addition, an investigation with a Geiger counter would show that a large proportion of the radioactive material deposited in the area of the cancerous tissue.

When in the method according to the invention the glycoprotein When brought into contact with neoplastic cell material, it causes this material to agglutinate and its agglomeration into lumps. This reaction distinguishes the neoplastic material from the normal Cell material whose cells remain isolated and not significantly agglutinated. The exact process after which this is currently unknown. It is not intended to be bound by any theory, however it is believed that the glycoprotein reacts with a substance essential for the cell membrane of the neoplastic Material specific and absent in normal cell membranes. The theory is put forward that

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a part of these substances consists of N-acetylglucosamine "which forms part of a larger molecule" and which can react specifically with the glycoprotein according to the invention and lead to the "observed agglutination". However, this reaction does not essentially take place if the glycopfotein comes into contact with normal cell material »Furthermore, if the glycoprotein used is labeled with a fluorescent, radioactive or visible dye, the corresponding fluorescence, radiation or color movement or characteristics can be determined on individual cancer cells. However, these phenomena would not be noticeable, if only normal cell material would be present in the test material.

example 1

The glycoprotein was extracted from wheat keinijbipase by the method described in an article "Identification of a Tumor Specific Determinant on Neoplastic Cell Surfaces" by MMBurger, AR Goldberg in "Proceedings of the National Academy of Sciences" 57s2, pp. 559-366, February 1967, is described. 10 g wheat germ were suspended in 500 ml of distilled water, ground in a mortar and then homogenized subsets ml each of 100 of the suspension was held for 15 minutes in a room kept at 63 ⁰ C water bath · These suspensions were then centrifuged und.das precipitant by decanting separated, but other separation methods can also be used. The liquid components obtained above were combined and clarified by being passed through a glass wool filter. 425 ml of this clarified liquid was cooled to ^{0 ° C.} This temperature was used to keep the ammonium sulfate solution saturated. The temperature may vary depending on the salt used in the salting out

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-π- 190305Q

can be varied so that the salt is kept in the state of saturation. 113g ammonium sulfate was added to the cooled clarified liquid. The resulting suspension was centrifuged for 20 minutes. The precipitate was separated and redissolved in 14 ml of water and then dialyzed in the cold. A precipitate formed during this dialysis was separated by centrifugation. The dialyzed fraction was then condensed by rotary evaporation (other condensation methods can also be used) at 4 ^° C. and passed through a polysaccharide gel column (Sephadex G-75), which was also ^{cooled to 4 °} C., and diluted with water. The glycoprotein was obtained in the drain after 300 to 375 ml of liquid was drained.

The purity of this product was determined in the "Spinco Model E" ultracentrifuge. The glycoprotein showed a single peak with a sedimentation coefficient of 2.74 at 2O ⁰ C in water. In addition, the product was subjected to electrophoresis on a foam rubber strip as a base by the method of MMBurger and! »Glaser, which is described in" Journal of Biological Chemistry "239, 3168 (1964). The glycoprotein was found to be pure and the extraction results were reproducible.

Example 2

The pure glycoprotein prepared according to Example 1

131

was treated with iodine 'marked as follows *. 25 mg of glycoprotein was mixed with water. To the mixture was added 1.25 mg of sodium carbonate. A Jodierungsgemisch was prepared by mixing 1.2 ml of a 0.002 molar sodium odidlösung, 1.2 ml of a 0.002 molar solution of sodium nitrite and 1.0 ml of iodine ^ prepared in sodium bisulfite. The iodine had an activity of 1 m0 / ml.

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190305Q

The Jodierungsgemisch was adjusted duroh addition of O, 5N hydrochloric acid to a _pH value in Section 4. The solution was then neutralized with 0.25N lithium hydroxide. The neutralized iodination mixture was diluted with 16.5 ml of water and added to the alkaline G-lyooprotein solution. The resulting mixture was poured onto a column of the ion exchange resin "Amberlite IR-4B". The effluent from the column, il the larger I _e containing the active labeled material - determined by standard infrared analysis -., Was then passed through a further exchange column of "Amberlite IR-IOOHf The product obtained from this second column was radioactive coded glycoproteins

Example 5

The G-lycoprotein according to the invention was labeled with a light isotope as follows: 100 ml of a glycoprotein solution containing 5% glycoprotein was reacted with 100 ml of a saturated sodium acetate solution. The mixture of glycoprotein and iosodium acetate was then cooled in an ice bath. Acetic anhydride marked with Hr (5 $ in acetone) was then added in five equal parts of 2 mg each over the course of 2 hours, while the temperature of the reaction mixture was kept at ⁰ ° C. The product obtained (acetylated glycoprotein) was then separated from the unreacted H -labeled acetic anhydride and - ^ acetate by dialysis against water.

Example 4

The glycoprotein of the invention was labeled with a light isotope as follows: 50 ml GIy cop fco te in solution containing Z ° J> glycoprotein in water was buffered with sodium bicarbonate to the preferred _pH value of 8 ». 5 The exact p ^ value and the special

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Buffer media are for the method according to the invention not crucially important. 5 ml of 0 - ^ - labeled acetic anhydride were added to the buffered G-Iyooprotein solution (156 in acetone). The solution was held at room temperature for about 10 hours "until the reaction was complete · The · O -labeled G-lycoprotein was then separated from the acetic acid and unreacted acetic anhydride using a Sephadex column, "until the larger rope of the biologically active material was eluted, determined according to the method in Example 2 described method.

Example 5

The glyeoprotein labeled with a fluorescent substance was prepared as follows: 10 ml of glycoprotein ($ 5 in water) were added with 10 ml of 0.05 molar Biocarbonate buffer (p ™ 8.5) implemented. This mixture was then with 10 ml of a 0.5 millimolar solution of H-acetylgluoosamine implemented. The suspension was with 3? Luorescein isothiocyanate on Kdaeelgur "Gelite" (manufacturer Calbioohem, Catalog # 34323) mixed. The GE the mixture was left to react for 2 hours. The eluorescein isothiooyanate glycoprotein was then from unreacted dye on "Celite" separated by centrifugation. The labeled product then became by distribution with the aid of a gel filtration column ("Sephadex") with water on the one described in Example 2 cleaned way.

Example 6
The glycoprotein according to the invention was labeled with radioopjR. ') Ceniiodine-125 ^w ^ ^{e as} follows: 500 mg of glycoprotein in 10 ml of salt buffer were with 12mC. Carrier-free iodine ¹²⁵ reacted by dropwise addition of the iodine, Naoh addition of 3 ml of water was added to the reaction mixture

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Until completion of the reaction left to room temperature and then dialyzed against water in the cold at 4 ⁰ C. The radio-opaque iodinated glycoprotein was obtained by conventional gel filtration,

Example 7

The glycoprotein according to the invention was labeled with a visible dye as follows: 500 mg glycoprotein were mixed with 1 mg sodium bicarbonate solution « The total volume was brought to 20 ml with water, 1 mg of 1-fluoro-2,5-dinitrobenzene in 20 ml of ethanol was added added. The resulting mixture was shaken at room temperature for about 2 hours. The mixture then became evaporated to dryness with removal of the ethanol and water. The residue was dissolved in water and extracted with alcohol. The visible glycoprotein was then redissolved in water.

Example 8

The glycoprotein was determined for its ability to differentiate neoplastic cells from normal cells, examined. Formal thymozjites from rats and mice were chosen for comparison with lymphoma tumor cells. Normal mouse thymocytes were obtained from the thymus of 5 week old Swiss mice prepared by harvesting in a phosphate buffered saline solution and after Isolated method described in "Cancer Research" 20, 1426

(1960) is described. Normal rat thymocytes were then extracted in "the same manner" from the thymus for 5 weeks get old-wistar rats. These normal cells were used as a comparative sample against malignant thymocytes. Malignant thymocytes (L ^ IO lymphoma cells) were transferred intraperitoneally in 6 week old Swiss white mice. After 7 days the Peritoneal fluid in phosphate buffered saline

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caught. The cells were then removed from the foreign cell material isolated and suspended in phosphate buffered saline and centrifuged for 5 minutes. The above Fluid was poured off and the cells resuspended in phosphate buffered saline (hereinafter referred to as PPS labeled) suspended, washed in PPS, whereupon the process was repeated. The cells were then again suspended in PPS and diluted with PPS to a concentration of 5 × 10 cells / ml. This diluted cell suspension was then combined with 0.02 ml of the glycoprotein in water (10 mg / ml) and at Left to stand at room temperature. A 0.05 ml drop of this solution was enclosed in a Sykes-Moore cell chamber and watched the hanging drop. The malignant thymocytes clustered to a degree of 3 to 4 on the following scale together, while normal cells either do not differ or are negligible Mass clumped together.

The following scale was used to measure the degree of clustering: 0 = no clustering}

. 1 = presence of only a few agglutinated cells during the majority of the remaining cells remain isolated; 2 = presence of a large number of small ones Agglutinations} 3 = all cells agglutinated in small groups j 4 = strong agglutination of all cells. The high degree of agglomeration that occurs in neoplastic Cells were easily observed and distinguished from the response seen in normal Cells occurred.

Example 9

Tissue culture fibroblasts were placed in tissue culture "spinners" bred in supplemented media. They were then transferred to PPS and concentrated with PPS diluted by 1x10 cells / ml * For comparison with these normal cells were treated with a preparation of

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term Ehrlich ascites hyperdiploid mouse fibroblasts on a 6 week old Swiss muse intraperitoneally Transferred These cells were obtained from the peritoneal fluid and suspended in PPS. The suspension was with PPS to a concentration of 1x10 Cells / ml diluted A suspension that contains any cell type was prepared in the manner described in Example 8. Examination under the microscope revealed that the neoplastic cells had a degree of aggregation 3 to 4 on the scale described in Example 8 showed, while the normal cells showed themselves either not clumped together or only so negligibly that a distinction between the two can be made Types of cells was possible.

Example 10

A blood sample was obtained from a patient suffering from leukemia using a modification of the Amos method, which is described in the report of the 9th Congress of the European Society of Hematology 1963, p. 1132. The blood sample was treated with a 5% solution of the disodium salt of ethylenediaminetetraacetic acid (Na ₂ EDiDA) and 5 % polyvinylpyrrolidine, which caused sedimentation of the red blood cells. The suspension was centrifuged and the above liquid was drawn off and diluted with a phosphate-buffered saline solution to a concentration of 5 × 10 cells / ml. A sample of normal blood was treated in the same way to obtain a leukocyte suspension "The two blood cell samples, one of which contained the normal leukocytes and the second the leukocytes from the leukemic blood, were treated as follows? A 0.3 ml sample of each cell suspension was treated with glycoprotein to a final concentration of 20 µg / ml.

One drop each of the suspensions obtained was then

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placed on a cover slip and enclosed in a Sykes-Moore cell chamber and observed for agglutination. It was found that the samples of the leukemic blood had a high degree of agglutination, as shown in the following table, while the suspension was from normal blood a relatively insignificant agglutination showed _β . This test thus clearly differentiates the neoplastic cells from the normal cells.

White blood cell specimen Relative degree of agglutination

Normal cells +

Leukemic cells +++

In this table, "O" means irrelevant Agglutination, "+" indicating less agglutinated Cells, while most of the cells remained isolated, "++" the presence of some small agglomerations of cells, "+++" agglomeration all cells present, and "++++" denotes massive agglomeration of all cells.

Example 11

A cell preparation was made from L2 lymphoma, 6C3HED lymphoma and TA3 adenocarcinoma. The L2 lymphoma was produced by irradiation according to the method of E. Shelton, which is described in "Journal of the National Cancer Institute", 12: 1203, (1952). The 603HED lymphoma was produced by reacting cells with estradiol benzoate according to the method of WeV.Gardner et al made in "Cancer Research": _4; described 73 (1944) the TA3 adenocarcinoma was taken from a naturally occurring mammary tumor in a female mouse of strain a the cell suspensions were prepared.. the cells by injection into the

to
Peritoneal cavity of a normal mouse / inject die

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Ascites sublimates were withdrawn and suspended in Tyrodes-Bffer, centrifuged downwards and then resuspended in 5% ethylenediaminetetraacetic acid and Tyrodes' buffer. A suspension of normal cells from the spleen, thymus and lungs of a mouse was prepared by removing the cell material from the associated connective tissue was separated by pressing the fragments through a fine sieve. The cell material obtained was then centrifuged and resuspended in the same manner as for the neoplastic cells. Normal liver cells were prepared in the same manner after the liver tissue was washed in situ in EDTA solution. Epidermal and bone marrow suspensions were prepared according to the method described in "Transplantation of Tissue and Cells", edited by ReE ₀ Billingham (Philadelphia: WistarJhsti7 tute Press 1961), pages 12 and 91. Since normal cells are generally smaller than tumor cells, the cell concentrations were adjusted so that cell surfaces of the same size were presented «

These concentrations were about 5 x 10 spleen cells /

ml of buffer versus 2 × 10 ⁶ L2 cells / ml of buffer. A 0.3 ml sample of each cell preparation was then with a suspension (10 mg / ml) of fluorodinitrobenzene-labeled G-lycoprotein, which had been obtained by the method described in Example 7, to a final concentration of 20 micrograms / ml treated. As the following table shows, the preparation containing the neoplastic cells agglutinated in each pail, and visible agglutinations were observed. In contrast to this, the preparations containing normal cells showed neither the discoloration by pluordlnitrobenzene nor the degree of agglutination which can be determined in neoplastic cells.

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(1 903050 ) 1 (1 reaction ) Observed (1 ⁾ +++ +++ -HH * O 0 0 0

Cell material

L2 lymphoma
603HED lymphoma
TA3 adenocarcinoma
Normal spleen cells

Normal thymus cells
Normal lung cells
Normal epidermal cells
Normal bone marrow cells
Normal liver cells +

(1) Visible agglomeration and simultaneous color reaction were observed.

The symbols used in this table have the same meaning as given in Example 10.

From these results it was concluded that the glycoprotein used in this test was valuable for the Distinction between normal and neoplastic materials is. The cells can in the method according to the invention can be used as a suspension in any conventional buffer media, e.g., Tyrodes buffer, "Versene", PPS and the like.

The glycoprotein according to the invention can be used for in vivo tests or in vitro tests to distinguish between neoplastic cells and normal cells. The concentration of the glycoprotein is not critically important and can be up to about 20% or more as required.

Example 12

L-1210 lymphoma cells were based on the method described above Way prepared and suspended. Each of these cell suspensions which has a concentration of 5x10 cells / ml, numerous solvents were added to determine their influence on the agglomeration reaction » To 0.3 ml of the cell suspension, which has a concentration

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tration of 5x10 cells / ml became glycoproteins (10 mg / ml in water) given in such an amount that the concentration was 40 micrograms / ml ". Like that Values in the following table show that the addition of the glycoprotein caused the cancer cells to agglomerate as a result, while agglomeration did not take place, if no glycoprotein was used

Summation 1 ung

1-1210 + & acetic acid 95 # MeOH 3-4 +

L-1210 + PPS '0 -

L-1210 + glycoprotein + 4 L-1210 + PPS 0

1-1210 10 $ ethanol + glycoprotein + 4 1-1210 10? 6 ethanol 0

L-1210 50 $ Ethanol + Glycopfcoifeein + 3

L-1210 $ 50 Ethanol 0-1

Example 13

Cancerous vaginal and urine cell material was obtained from patients which were known to have cancer. These cells were also suspended and to a concentration of about 5x10 cells / ml diluted. Glycoprotein was added to a 0.3 ml sample to a concentration of 50 micrograms / ml, to determine the agglutinating effect «As the following table shows, found in every pail in which the Glycoprotein was added, a strong agglomeration of cells in contrast to the cell material that took place suspended but not treated with glycoprotein.

Agglomeration vaginal + PPS 0

Vaginal cells + PPS + glycoprotein +2 urine cells + PPS 0

Urine cells + PPS + glycoprotein + 2-3

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-27- 19030

Example 14

Cancerous kidney cells from urinary embryos maintained in Hanks-Eagle medium were warmed to 37 ° C and _{treated with 0.2 ml of 2 tons of} 5 # trypsin separated, were. The suspensions were then aspirated, condensed, centrifuged, washed with PPS, buffered and diluted to a concentration of 5 x 10 cells / ml. A 0.3 ml sample of the cell suspension was treated with glycoprotein to a final concentration of 20 micrograms / ml _{The cell suspension showed a degree of agglomeration of 4 on the scale e} explained in the preceding examples. The untreated sample did not show any agglomeration.

Example 15

HeLa cells maintained in Hanks-Eagle solution were raised isolated and diluted in the manner described in Example 14 ". A 0.3 ml sample of the cell suspension was replenished with G-lyooprotein to a final concentration of 20 micrograms / ml treated «The cell suspension showed a degree of agglomeration of 4 on that discussed above Scale · The untreated sample did not show any agglomeration.

As already mentioned, the glycoprotein according to the invention can be used for an in vivo or in vitro test method can be used to distinguish between neoplastic cells and normal cells. The concentration of the The glycoprotein used is not critically important and can be up to about $ 20 or more, like the Circumstances require. · If the test is performed in vivo the glycoprotein can be administered by any convenient route, for example topically, parenteral, oral, rectal and vaginal. The invention is for the detection of neoplastic cells in mice,

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Rats, dogs, cats and other warm blooded animals including of humans, where the localization is for experimental, diagnostic or clinical Purposes is necessary. For this purpose the labeled or unlabeled glycoprotein is then converted into the usual pharmaceutical ! Carriers can be incorporated, e.g. in sterile buffered isotonic media, aqueous suspensions, syrups and solid preparations, e.g. tablets and capsules, such as them. in a compendium, e.g. in Husa's Pharmaceutical Dispensing and Remington's Practice of Pharmacy.

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Claims (12)

Claims 1) Procedure for the detection and localization of neoplastic cell material, characterized in that that the cell material is brought together with a glycoprotein which is responsible for the specific binding of the G-lyooprotein leads to neoplastic cells and has been previously marked in such a way that a reaction can be determined is. 2) Method according to claim 1, characterized in that that the cell material is brought together with an effective agglutinating amount of a glcoprotein, that has been extracted from wheat germ and the cell material is observed for a detectable reaction, which takes place when neoplastic cells are present. 3) Method according to claim 2, characterized in that the detectable reaction is a visible and " is measurable fluorescence. 4) Method according to claim 2, characterized in that the detectable reaction is the presence of a visible dye on the neoplastic material. 5) The method according to claim 2, characterized in that the detectable and detectable reaction Emission of radiation by the neoplastic material is. 6) The method according to claim 2, characterized in that an effective amount of the G! Fecoprotein is given to a patient administered and observed for a predetermined response. 7) Method according to claim 6, characterized in that the predetermined response is the localized emission of radiation 908839/0925 8) Method according to claim 6, characterized in that the predetermined reaction is a radio-opaque material determines with the X-ray screen or pluoroscope, 9) Glycoprotein, which is suitable for the detection and identification of neoplastic cells, thereby marked that it a) has the following properties: a maximum optical density of 278 mi, a minimum optical density of 252 nm., a sedimentation coefficient of 2.74 in water "at 20 ⁰ C, a diffusion coefficient of 9.2 χ 10 *"'cm / Sec., A partial specific volume (from oycnometry) of 0.71 ml / g »a partial specific volume of 0.72 ml / g from the amino acid composition, a maximum viscosity of 8 ms 10 ml / cm second", an approximate one Molecular weight of 26,000, a friction ratio of 1.2, an axial ratio of 4 Ms 5, an intrinsic viscosity of about 9 at 25 C, an amino acid composition shown in Table I, and a carbohydrate content ($ 3-5) that is almost exclusively all glucose units, and b) has been marked beforehand in such a way that the determination a reaction is possible after the neoplastic cells merged with the glyeoprotein have been. 10) glycoprotein according to claim 9, characterized in that it is associated with a fluorescence-generating compound is marked. ■ 11) glycoprotein according to claim 9, characterized in that it is marked with a visible dye. 909839/0925 12) glyooprotein naoh claim 9, characterized in that that it is marked with a radioactive material » 909839/0925 Blank page