LU82345A1 - CANCER DETECTION METHOD - Google Patents

Description

; OC 51.100

8 0-¾ Λ * rt GRAND-DUCHY OF LUXEMBOURG

from 8 ...... April ..... 19.8ç> ________________ Dear Minister ____, öSsäb of National Economy and the Middle Classes

Book title: ........................................

cÿvWÎM Industrial Property Service

LUXEMBOURG

Invention Patent Application I. Request .... La .... soa ± êfcé ...,. & Ifc £ ..ï BRISTOL.-M.y.EES ..... COMEÂN.X.i! ........................ (1) .... â .... liEKr.YQBK./......N.# .y "...... 1.0.022 / ..... Efcatsrünis ..... d ^ êrigue * ..... represented ................ ......

, by Monsieur Jacques ..... de Muyser * ...... acting as inan -.......... (2) ..... dâ.tai.re ... .............'.................................... .................................................. .................................................. .................................................. ...............................................

drop this .... tolfc ._ .. a ^ ..................................... ....................................... (3) to ....... .15 ............. .... hours, at the Ministry of National Economy and the Middle Classes, in Luxembourg: 1, this request for obtaining a patent d invention relating to:> ..... "Method ..... of ..... detection of cancer" ....................... .................................................. .................................................. ..... {4) ........................................

.................................................. .................................................. .................................................. .................................................. .................................................. ............................. (5) 2. the delegation of power, dated ÎÏ.EW “. Y.QBÆv .................................... on .4. April 198o .................

3. description in French ......... language .................................. of the invention in two copies; 4 ........ /. / ............... ... drawing boards, in two copies; 5. the receipt of the taxes paid to the Luxembourg Registration Office on ...... 8 ..... April 198o ................... .................................................. .................................................. .................................................. ..........................................

claims for the above patent application the priority of one (or more) application (s) of (6) .................. patent ........ .......................................... filed (s / dn (7) in the United States of America .................

le ...... lo ... avril ..... 19.79 __________ (No ...... 028.732) ____________ (8) r on behalf of S inventors ........... .................................................. .................................................. .................................................. ........................... (9) elect domicile for him / her and, if designated, for their proxy, in Luxembourg .. ...................................

...... 25 / ..... blïïr ~ Rôvâl .................................. .................................................. ... (i0) requests the issuance of a> invention patent for the subject described and represented in the appendices

Ektiκτηrationnées, - with postponement of this issue to ....... 5 ................................. .....month.

mandaj | airè .............. δ ........... V V

Π. Deposit Minutes

The aforementioned patent application has been filed with the Ministry of National Economy and the Middle Classes, Industrial Property Service in Luxembourg, dated: April 8, 198o ........ ·· .. Pr. The Minister to .......... 15. hours λ of the National Economy and the Middle Classes, / p-d.

P * ·. ; . g '·} |> -7 ί, s # / D. 51.100

CLAIM OF PRIORITY

OF THE PATENT APPLICATION / / & \ TO THE UNITED STATES OF AMERICA FROM APRIL 1979 Brief Description filed in support of an application for

PATENT

at

Luxembourg

in the name of: BRISTOL-MYERS COMPANY

Λ p0tir: "Cancer detection method".

*

The present invention relates to a diagnostic test method for the detection of malignant tumors.

New methods are undoubtedly necessary for the detection of cancer. In many 5 cases, the early diagnosis of cancer would greatly increase the chances of complete remission of the disease.

The literature describes attempts that have been made to demonstrate the presence of tumor specific components such as hormones and antigens in the blood of cancer patients. However, these attempts have largely failed and a practical, non-aggressive diagnostic method based on the level of a specific component of the tumor in the serum is a goal that has so far been unachievable.

15 Recently, the presence of DNA-binding proteins

has been detected in the serum of patients with neoplasia, systemic lupus erythematosus and other inflammatory disorders (see, for example, FEBS Letters 92 (2): 211-213 (1978) j Europ J. Biochem. 71 1-8 (1976); 20 Amer. J. Med. 65: 437-445 (1978)]. However, no correct analytical method, which would be necessary for a practical diagnosis, has been proposed for the search for these serum proteins In addition, the serum proteins described in the prior art did not appear to exhibit the desired degree of selectivity in a cancer screening method.

The serum inhibitory protein used in the present invention is described in The Pharmacologists - (Abs.) 20 (3): 238 (1978). However, the summary does not give any indication of the presence of protein at different rates in cancer patients compared to non-cancer patients.

The present invention provides a diagnostic test method generally applicable to the detection of malignant tumors in humans. More particularly, the invention provides a diagnostic method indicating the existence of malignant tumors by analysis of human serum samples to detect the presence * 2 "? of a specific DNA-binding protein. The method is useful for the early detection of a malignant disease and as an indicator of disease progression in a cancer patient under treatment.

The method of the invention is based on the observation that a specific DNA-binding protein (hereinafter called inhibitory protein X) is present at significantly reduced concentrations in the serum of comparatively cancer patients to non-cancer patients. By providing a sensitive test for these serum proteins, the present invention allows rapid, non-aggressive and precise detection of the existence of cancer.

The presence of a particular DNA-binding protein in human serum has been demonstrated in parallel with the present invention in the course of research relating to the bleomycin-induced degradation of PM-2 DNA. (Circular DNA covalently closed, from Pseudomonas bacteriophage). The observation of the inhibition of this degradation by human serum opened the way to studies on the nature of the inhibition and to attempts to identify the inhibitor compound.

The Applicant's hypothesis that the inhibition of the PM-2 DNA degradation reaction is due to a serum protein was confirmed when treatment with pronase resulted in a decrease in inhibitory activity. Treatment with DNAse I or pancreatic RNase has no effect on the inhibitory activity of serum. Dog and calf sera have not been shown to be inhibitory, nor have albumin and human immunoglobulins.

The inhibitory protein was purified under non-denaturing conditions by the use of molecular filters, dialysis and column chromatography of "Sephadex". The purification of the protein was considerably improved by the binding of the protein to the DNA, the isolation of the DNA-protein complex from the unbound protein and then the dissociation of the protein from the DNA by treatment with 3 <- 1 'urea. Purification by SDS-polyacrylamide gel electrophoresis and by isoelectric point precipitation is considered to give a degree of purity more than 2000 times greater. Gel electrophoresis with isoelectric precipitation indicates that the inhibitor is a protein whose molecular weight approaches 64,000 daltons and whose pi is equal to 5.9 * This protein has been provisionally called protein inhibitory X in the present specification.

Inhibitory protein X exerts its inhibitory effect by binding to DNA. DNA binding has been demonstrated by agarose gel electrophoresis, isolation of the DNA-protein complex, quenching of fluorescence and studies of circular dichroism.

In the study of the nature of the protein inhibitor X, it was discovered the very unexpected fact that there is a notable difference between the level of this protein in the serum of cancer patients and its level in the serum of non-cancer patients. From this important discovery, an attempt was made to establish a sensitive and rapid method for detecting the presence of the inhibitory protein (X in human serum.

PM-2 DNA has already been used in a spectrofluorometric method to determine the biochemical activity of bleomycin (Cancer Res. 38 ï 3322-3326 (1978)). The mechanism of action of bleomycin appears to be related to its ability to degrade DNA, and the decrease in the binding of ethidium bromide (2,7-diamino-10-ethyl-9-phenylphenanthridinium bromide) to bleomycin-induced PM-2 DNA, as determined by fluorescence spectrometry, can therefore be used to assay bleomycin activity.

As indicated above, the inhibitory protein X has been observed to act as an inhibitor of the degradation of PM-2 DNA under the effect of bleomycin. For this reason, the fluorometric test used in the prior art to measure the activity of bleomycin can also be used to determine the concentration of protein 1. 4

S

inhibitor X in human serum. The level of inhibitory protein X can then be used to predict the probability of cancer in the patient being examined.

The present invention therefore provides a method of detecting cancer in humans, which comprises the following steps: (a) taking a blood sample from a patient examined for cancer; (B) determining the concentration of inhibitory protein X in this sample; and (c) comparing the concentration determined in step (b) with the concentration of normal inhibitory protein X corresponding to the serum of non-cancer patients, a significant reduction in the concentration of inhibitory protein X compared to the normal concentration being a cancer proba-20 index.

The particular assay method used in step (b) to determine the concentration of inhibitory protein X in serum is not critical, and the invention, in its broadest sense, is that it is established that the concentration of inhibitory protein X (regardless of how it is determined) can be used as a diagnostic means for a wide variety of human cancers.

One titration method which has been found to be advantageous is the PM-2 DNA fluorometric method described in Cancer Res. 38: 3322-3326 (1976). The use of this method is based on the inhibitory effect of the inhibitory protein X on the well known bleomomycin-induced breakdown of the ADM response of 35 PM-2. When this analytical method is used, it is advantageous to express the inhibitory protein X concentration by a CI ™ value defined as being the inhibitory protein X concentration required to inhibit 50% of DNA degradation. bleomycin-induced PM-2. This CI ^ q value can then be compared to the normal CI ^ q value corresponding to the serum of non-cancer patients, and a significant increase in the CI ^ q value from the normal value is an indication of probable cancer. .

Details on the method of fluorescence assay of PM-2 DNA of the inhibitory protein X are given below: 10 A mixture of a serum sample (for example a 50 μl sample) is first prepared £) of a patient to be examined with a solution of bleomycin, PM-2 DNA and 2-mercapto-ethanol in a buffer at pH 9> 5- Normally, a sample of 50 y5, of serum is mixed with 450 μl of a solution containing 35 nanomoles of bleomycin, 8.3 micromoles of PM-2 DNA and 25 millimoles of 2-mercaptoethanol in a sodium borate buffer of pH 9.5 (0.015 M NaCl: 0.05 M sodium borate).

The above mixture is then incubated at 37 ° C for 30 minutes.

A solution of ethidium bromide in a denaturation buffer of pH 12.1 is then prepared by adding 0.1 ml of a solution of ethidium bromide (22 μg of ethidium bromide per ml of denaturation buffer of pH

12.1) to 0.9 ml of denaturing buffer of pH 12.1. A suitable denaturation buffer is a 0.09M Na ^ PO ^ buffer: 0.01M EDTA - 0.01M NaCl, pH adjusted to 12.1 by addition of 0.15M NaOH. To 1 ml of this ethidium bromide solution is added an aliquot (for example 100 µl) of the incubated serum mixture, prepared as indicated above.

The fluorescence of the mixture of ethidium bromide and PM-2 DNA is determined using a spectrophoto-fluorometer for an excitation of 530 nm and an emission of 35 590 nm. Fluorescence higher than the background is due to the binding of ethidium bromide to PM-2 DNA, and a variation in fluorescence compared to a control reaction (identical to the sample to be analyzed, but not containing * 6 bleomycin) quantitatively expresses the degree of degradation of PM-2 DNA under the influence of bleomycin. From the fluorescence values, it is easy to determine the percentage of inhibition of the degradation of PM-2 DNA due to the presence of the inhibitory protein X in the serum.

The concentration of inhibitory protein X in the serum sample is then determined by the standard Lowry method (Polin phenolic reagent), as described in J. Biol. Chem. 193: 265-275 (1951).

The graphical representation of the percentage inhibition of degradation of PM-2 DNA as a function of the logarithm of the concentration of inhibitory protein X makes it easy to determine the value CI ^ q for the particular serum sample.

Once the CI ^ q value has been calculated for a given patient, this value can be used directly to predict the probability of cancer in that patient. Extensive studies in healthy (non-cancerous) patients and patients with a wide variety of malignancies have shown that inhibitory protein X is found in significantly lower amounts in the serum of cancer patients. Consequently, the CI5Q value for cancer patients is found to be notably high compared to the normal value corresponding to healthy subjects. This clear differentiation of the CI ^ q values provides a simple and precise basis for detecting the presence of cancer and forms the main object of the present invention.

The precise CI ^ q value obtained from blood samples from 30 non-cancer patients varies to some degree with the particular subject. In the patients examined to date, the mean CI5Q value for non-cancer patients has been found to be 90.2 + 11.0 yg / ml (significance level: 0.001). The highest IC 50 value observed for a healthy patient was 120 µg / ml.

In cancer patients, the CI ^ q values obtained vary with the type of cancer involved, but in all cases known so far, the value has been "* 7 significantly higher than that obtained for" subjects not cancerous. The lowest CI ^ q value known to date for a cancer patient has been 270 yg / ml. However, in general, a CI ^ q value greater than about 5,200 indicates that the patient is most likely to have a malignant tumor of one type or another.

The method of the present invention has been used successfully to detect a wide variety of types of cancer and is therefore considered to apply generally to the diagnosis of cancer. As specific examples of the types of tumors detected, there may be mentioned testicular cancer, lymphoma, leukemia, breast adenocarcinoma, small and large lung cell adenocarcinoma, colon adenocarcinoma and melanoma, 15 to only cite a few. The method of the invention also appears to be specific for the diagnosis of cancer, since non-cancer patients with various kinds of non-neoplastic diseases have not exhibited high IC ^ q values associated with the presence of cancer.

The fluorescence test described above for the search for the inhibitory protein X according to the present invention is based on an intrinsic property of this substance, namely the inhibition of the degradation of the DNA of PM-2 induced by bleomycin. Levels of this protein in serum, determined by this method, have been correlated with the presence or absence of a malignant disease in the present invention. Having a purified inhibitory protein X makes it possible to use for its assay a variety of other methods known in the field of biochemistry and clinical chemistry. Among these methods, there are mainly immunological methods such as the radioimmunoassay, the fluoroimmunoassay, the enzyme immunoassay (for example ELISA), the spin immunoassay, the immunoassay of chemiluminescence, the fluorescence polarization immunoassay, the nephelometric test, gel diffusion methods and 8 conventional methods such as hemagglutination and complement fixation. In addition, other functional properties of the inhibitory protein X can be exploited for its determination in biological fluids. Catalytic phenomena associated with the molecule can be measured in spectrophotometric or spec trofluorometric kinetic tests or based on a turning point. In addition, separation technology can be applied to the liquid in question, and various detection methods can be used to quantitatively assay the protein, for example spectral or densitometric methods and immunological methods. Whereas the above methods specifically determine the concentration of inhibitory protein X, it follows that serum levels of the protein determined by such methods are correlated with the presence or absence of a malignant disease .

The inhibitory protein X was purified according to the procedures below.

20 Partial purification - Procedure A.

The inhibitory protein X is partially purified by chromatography on a "Sephadex" column. 11 ml of human serum are loaded onto a column of "Sephadex G-75" (88 cm x 2 cm), then the elution is carried out in a sodium phosphate buffer (0.07 M, pH 6, 8) at a flow rate of 6 to 8 ml per hour, at room temperature. The inhibitor is contained in the volumes of voids which are collected, lyophilized, resuspended in 1 ml of water, dialyzed against 20 volumes of water for 24 hours at 4 ° C., then loaded onto a column of "Sephadex G-200" balanced in the same buffer.

The elution from the column of "Sephadex G-200" gives three peaks which absorb at 280 nm. Peak III which represents 7.7% of the total protein eluted contains the inhibitory protein X. This fraction is treated as described above, then loaded again on a column of "Sephadex G-200". Elution is carried out with the phosphate buffer described above. We collect fractions of 9

3 ml of each column using a fraction collector (Gilson Medical Electronics, Inc., Middleton, Wis.), And the elution curves are plotted by measuring the absorption at 280 nm using a spectrophotometer. Protein concentrations are determined by the Lowry method. Purification - Procedure B

The purification of the inhibitory protein is carried out by molecular filtration using "Amicon" filters, by dialysis and by column chromatography on a gel of "Sephadex". Human sera (1 to 5 ml) are centrifuged in conical "Amicon" filter tubes (Amicon, Lexington, Mass.) At 5000 x g for 10 minutes at 4 ° C. The retained portion which remains in the filter cone after centrifugation is resuspended in deionized water, the volume of which is adjusted to the initial volume of serum. Aliquots of the residue and the filtrate are tested for inhibitory activity. The active residue is dialyzed in a cellulose tube against 20 volumes of deionized water at 4 ° C for 24 hours. The residue and the dialysate are lyophilized and resuspended in deionized water. The results of the tests on the two fractions show that the inhibitory activity resides in the portion retained. This portion (1 ml) is loaded on a 60 x 5 cm column of "Sephadex G-50" and eluted with a sodium phosphate buffer (0.07 M, pH 6.8) at a flow rate of 30 ml per hour at 4 ° C. The column eluate (5 ml per tube) is collected using a "Buehler Fractomette Alpha 200" fraction collector (Searle, Fort Lee,. N.J.). The absorption at 280 nm of each fraction is measured with a spectrophotometer and the tubes are assembled in accordance with the absorption peaks. The combined fractions are lyophilized, dialyzed and the inhibitory activity is determined by the method described above. The "active" substance contained in the volume fraction of 35 voids is loaded onto "Sephadex G-75" and "G-100" columns and eluted in the same way as the "G-50" column. inhibitor is found in the void volume fractions of the two columns The "active" substance which was collected in the void volume fraction of the Sephadex column "G-200" is eluted with phosphate buffer sodium (0.07 M, pH 6.8) at room temperature, at a rate of 6 to 8 ml per hour. Fractions 5 are collected (3 ml per tube) with a “Gilson” micro-fraction collector (Gilson, Middleton, Wis.). The column eluate is fractionated on the basis of the absorption profile of 280 nm by collection of tubes below each absorption peak. Each fraction is then lyophilized, dialyzed and the inhibitory activity is determined as described above. The inhibitory activity is found in fraction III. The substance contained in the fraction III is loaded again on the Sephadex ”G-200” column and eluted under the same conditions. Fraction III of column 15 Sephadex "G-200" containing the inhibitory protein X represents a purification factor greater than 1000.

The method of fluorescence analysis of PM-2 DNA to assay the inhibitory protein X is described in detail below: Determination of the value CI ^ q for the inhibitory protein X.

The determination of the CIcn value (concentration of the protein necessary to inhibit the degradation of PM-2 DNA by 50%) for assaying the inhibitory protein X 25 in human sera is carried out in two steps: 1. The protein concentration in the serum is determined by the Lowry method.

2. The use of the PM-2 DNA fluorescence test for bleomycin, so as to obtain a dose response curve for the serum protein inhibitor. The Cl ^ g values are determined from logarithmic graphical probit representations of the variation in the percentage of inhibition of DNA degradation as a function of protein concentrations.

,. ‘11, · Operating modes; I. Lowry reaction * A. Preparation of stock solutions:

Solution A. 2% Na ^ CO ^ in decinormal sodium hydroxide.

Solution B. CuS0 ^ .5H20 at 1% in water.

Solution C. Double sodium and potassium tartrate 2% in water.

Solution D. Phenolic reagent of Folin- 1 ° Ciocalteau 2N.

B. Preparation of reagents in solution Reagent in solution 1. Mix 1 volume of stock solution B with 1 volume of stock solution C.

15 Reagent in solution 2. Mix 50 volumes of stock solution A with 1 volume of reagent in solution 1.

Reagent in solution 3. Mix 1 volume of stock solution D with 1 volume of water.

20 C. Reactions 1. Dilute the sera 1:10 with water.

2. Transfer 10 μΐ of the diluted sera to a 13 x 100 mm glass test tube and add 290 μΐ of water.

3. Add 1 ml of reagent in solution 2, mix well and incubate at room temperature for 10 minutes.

4. Add 100 μΐ of reagent in solution 3, shake well and incubate for 30 minutes, at room temperature.

5. Use a spectrophotometer to measure the absorption at 750 nm.

6. Determine the protein concentration from the calibration curve for beef serum albumin.

D. Calibration curve for beef serum albumin (SAB). 1. Prepare serial dilutions of SAB (eg 25, 50, 75, 150 pg / ml in a final volume of 0.3 ml.

2. Carry out the reactions indicated in part C, starting from step 3 (addition of the reagent in solution 2).

, ‘12. 3 · Plot the variation in absorption at 750 nm as a function of the SAB concentrations.

II. Protein A inhibitor assay Perform serial dilutions of serum protein 5, based on serum protein concentrations determined by the Lowry reaction. Perform dilutions in a constant volume of buffer. with sodium borate (0.05 M, pH 9.5).

B. Add the serum protein samples (volume 10 50 μΐ) to 10 μΐ bleomycin (34.5 nanomoles) contained in a glass test tube (13 x 100 mm).

C. Add 390 μΐ of sodium borate buffer (0.05 M, pH 9.5) containing 25 mM 2-mercapto-ethanol.

D. Add 50 μΐ of PM-2 DNA (8.3 x 10 "6 M in 0.15 M sodium chloride).

E. Incubate for 30 minutes at 37 ° C.

F. Transfer aliquots in triplicate (100 μΐ) to glass test tubes (13 x 100 mm) containing 0.9 ml of Na ^ PO ^ (0.09 M, pH 12.1) containing 20 of 0.01 M EDTA and 0.01 M sodium chloride

Shake well.

G. Add 100 μΐ of ethidium bromide (55.7 x 10 ”^ M) to the trisodium phosphate buffer, pH 12.1. Shake well.

25 H. Witnesses: 1. Sodium borate (0.5 M, pH 9.5) containing 2-mercapto-ethanol without bleomycin and proteins.

2. Protein without bleomycin or PM-2 DNA.

3. Protein without bleomycin.

4. Sodium borate (0.05 H, pH 9.5) containing a blank of 2-mercapto-ethanol.

I. Spectrophotofluorometry (Aminco-Bowman)

Transfer the sample to a small 1 cm quartz cell and measure the fluorescence for an excitation of 530 nm and an emission of 590 nm.

Claims (3)

1. Method for detecting cancer in human beings, characterized in that it consists of: (a) taking a blood sample from a patient undergoing cancer screening; (b) determining the concentration of inhibitory protein X in said sample; and (e) comparing the concentration determined in step (b) with the normal concentration of inhibitory protein X associated with the serum of non-cancer patients, a significant reduction in the concentration of inhibitory protein X relative to the concentration normal being the index of the probability of cancer. 2. Method for detecting cancer in human beings, characterized in that it consists of: (a) taking a blood sample from a patient subjected to cancer screening; (b) determining in said sample the concentration of inhibitory protein X necessary to inhibit 50% of the degradation of the PM-2 DNA induced by bleomycin j and | (c) comparing the CI ^ q value determined in step (b) with the normal CI ^ 0 value associated with the serum of non-cancer patients, a significant increase in the ClgQ value from the normal value being the index of the probability of cancer. 3- Method according to claim 2, characterized in that the determination of the value CI ^ 0 in step (b) is carried out by the following steps: (1) preparation of a mixture of the blood sample with a solution of bleomycin, PM-2 DNA and 2-mercapto-ethanol in a buffer of pH 9.5; (2) incubation of the mixture from step (1) for 30 minutes at a temperature of 37 ° C; (3) adding an aliquot of the mixture from step (2) to a mixture of ethidium bromide in denaturing buffer of pH 12.1; * * 14. * 'i' «· (4) determination of the fluorescence of the mixture of ethidium bromide and DNA from.PM-2 of step (3) with a = - spectrophotofluorometer for excitation at 530 nm and emission at 590 nm; 5 (5) determining the percentage of inhibition of bleomycin-induced degradation of PM-2 DNA based on the variation in fluorescence of the sample compared to a control sample not containing bleomycin; (6) determination of the inhibitory protein X concentration by the Lowry method; and (7) determining, from the values obtained in steps (5) and (6), the concentration of inhibitory protein X necessary to inhibit 50% of the degradation, induced by bleomycin, of the DNA of PM-2. !