GB2142428A - Adenocarcinoma related antigenic determinants and antibodies specific thereto - Google Patents

Abstract

A method suitable for use in diagnosing a human adenocarcinoma by determining adenocarcinoma-related antigenic material in a sample taken from a human patient comprises using (a) anti-K antibody or (b) anti-P<k> antibody to determine material in the sample having, respectively, (a') exclusively those antigenic determinants K specific to adenocarcinomas or (b') those antigenic determinants p<k> associated with the K determinants on the adenocarcinomas. Also disclosed are conjugates of anti-K and anti-P<k> antibodies with anti-tumour drugs or cytotoxic agents.

Description

SPECIFICATION Adenocarcinoma related new antigenic determinants and antibodies specific thereto, processes for their preparation and reagent systems pertaining to them The present invention refers to adenocarcinoma related new antigenic determinants, to antibodies specific thereto and to methods for the diagnosis of human adenocarcinomas based upon demonstration of said antigenic determinants in biological fluids, in tissue extracts or sections and in vivo on the tumor cells or masses.

The invention also concerns the use of specific antibodies in the therapy of adenocarcinomas for conveying anti-tumor drugs or other cytotoxic agents to the tumor cells or masses. It is known that in case of tumor pathologies it is possible to isolate, from the tumor masses themselves or from the biological fluids, substances associated with said pathologies. Many of these substances have antigenic properties more in the animals than in the man; some of them, generally named "tumor markers", are oncofetal antigens.

The presence of an antigen which is stated to be mainly associated with adenocarcinomas, and and particularly with their metastases, has been demonstrated by Gold et al, J. Expt. Med. 121: 439-462 (1965).

This antigen, known as carcinoembryonic antigen (CEA) can be assayed both in biological fluids and in tissue extracts or sections by various known methodologies, e.g. those reported in U.S. patents No.

3,663,684 and No. 3,697,638; Ann. of Clin. and Lab. Science 4, 5,357, 1974; Clin. Chem. 19, 10, 1973; Cancer 34:1504-1509, 1974; Clin. Res. 19:143, 1971; Proc. Natl. Acad. Sci. USA 64:161-167, 1969; Cancer 37:62-81, 1976; Scand. J. Immunol., Vol.7 Suppl. 6., 1978; Cancer45:1243-1247, 1980; and J. Natl. Cancer Inst. 57, 1, 1976.

The numerous studies on this matter including, e.g., the above references, have however revealed the very low specificity of CEA as this has been demonstrated not only in case of tumoral pathologies but also in presence of different, i.e. non tumoral, pathologies such as, e.g., many degenerative-inflammatory processes, and even in healthy donors. Moreover, biochemicaol studies [Urba R., Awl pert E., Isselbacher K.J., Proc. Natl. Acad. Sci. U.S.A. 72,4602-6(1975)1 have have shown differences between CEAs obtained from different laboratories, with consequently different clinical results obtained when the same techniques are used with materials of different origin.

It has now been found that it is possible to obtain from human adenocarcinoma metastases, as well as from all human adenocarcinomas, e.g. gastric, mammary and thyroid adenocarcinomas, antigenic determinants (K) which are "specific" to adenocarcinomas and antigenic determinants (pk) which are "associated" with the K determinants on the adenocarcinomas. While the pk antigenic determinants show a very strong cross-reaction with the antigenic determinants (P") which are present in plasma or serum or normal donors, the K antigenic determinants do not show any cross-reaction with the P" antigenic determinants. The mixture of said antigenic determinants K and pk and, perhaps, of some other unknown antigens, is what was previously known as CEA.

By using the above mentioned antigenic determinants K and antigenic determinants pk and P", specific antibodies anti-K, anti-Pk and anti-P" are obtained, according to the invention.

The use of the antigenic determinants K and pk and of the antibodies anti-K and anti.Pk specific thereto in the known immunological methods, such as, for instance, radioimmunoassay, enzymeimmunoassay, immunofluorescence, immunoluminescence, and many others, allows to detect and quantify exclusively those antigenic determinants Kwhich are specific to the adenocarcinomas, without interference deriving from the antigenic determinants P" of the normal plasma or serum, or, if desired, those antigenic determinants pk which are associated with the K determinants on the adenocarcinomas. The above determination may be carried out either qualitatively or quantitatively or semi-quantitatively.

The present invention provides therefore an improved method for the diagnosis of human adenocarcino mas through the determination of adenocarcinoma related antigenic material, either in a sample taken from a human patient, e.g. in vitro on biological fluids or tissue extracts or sections, or in vivo on the tumor cells or masses, wherein the improvement consists in determining, by means of specific anti-K or anti-Pk antibodies, exclusively those antigenic determinants K which are specific to adenocarcinomas without revealing the P" antigenic determinants of the normal cells, or, respectively, those antigenic determinants pk which are associated with the K determinants on the adenocarcinomas. So false positive results are reduced.

The expression "antigenic determinants K" refers to antigenic determinants which, as already said, are specific to the adenocarcinomas and which, showing no cross-reaction with the P" antigenic determinants of the normal cells, are recognized only by antibodies specific to them, i.e. anti-K antibodies: this definition includes any CEA antigenic mixture wherein the antigenic determinants other than the K ones have been totally or substantially removed.

The expression "antigenic determinants pk" refers to antigenic determinants which are present on the adenocarcinoma cells in "association" with, i.e., together with, the antigenic determinants K specific to the adenocarcinomas, the said pk antigentic determinants showing, as already said, cross-reaction with the P" antigenic determinants of the normal cells: this definition includes any CEA antigenic mixture wherein the antigenic determinants other than the pk ones have been totally or substantially removed.

The P" antigenic determinants are, as already said, antigenic determinants present in the normal plasma or serum and cross-reacting with the pk antigenic determinants.

An anti-K antibody is either an antibody obtained immunizing animals with the above said K antigenic determinants, or any anti-CEA antibody (i.e. the antibody produced immunizing animals with the CEA) modified in such a way that the antibody sites other than the anti-K ones have been therein totally or substantially saturated by antigenic determinants specific to them.

Similarly, an anti-Pk antibody is either an antibody obtained immunizing animals with the above said pk antigenic determinants, or any anti-CEA antibody modifed in such a way that the antibody sites other than the anti.Pk ones have been therein totally or substantially saturated by antigenic determinants specific to them.

Anywhere in this specification the term antibody(ies) refers both to immunoglobulins and to immunoglobulin fragments and may also mean the corresponding antiserum.

Furthermore, unless otherwise indicated, the term antibody means a polyclonal antibody; when a monoclonal antibody is intended, this is specified.

Two alternative processes are provided by the present invention for preparing anti-K antibodies specific to the K antigenic determinants.

According to one process anti-K antibodies are obtained by absorbing antibodies produced against a mixture of K and pk antigenic determinants with P" or pk antigenic determinants so as to saturate the pk immunological binding sites on the said antibodies.

According to the other process anti-K antibodies are obtained by: 1) immunizing animals with P" or pk antigenic determinants to produce antigen or anti-Pk antibodies; 2) purifying a mixture of antigenic determinants K and pk by means of the antigen or anti-Pk antibodies produced according to 1), so obtaining K antigenic determinants; and 3) immunizing animals with K antigenic determinants obtained according to 2).

The scope of the invention includes, in addition to the anti-K specific antibodies obtained by the two above alternative procedures, also the K antigenic determinants produced according to step 2) of the second alternative method described above, and, furthermore, the pk and P" antigenic determinants and the anti-Pk and antigen antibodies specific thereto, which are involved in the above processes.

Also the labelled forms, e.g. radiolabelled, enzyme-labelled, or fluorescence-labelled forms, of the hereabove said K, pk and P" antlgenic determinants and of the corresponding anti-K, anti-Pk and antigen antibodies, are included within the scope of the present invention.

In accordance with the first hereinbefore described method for preparing anti-K antibodies, the mixture of antigenic determinants K and pk iS injected in an animal, e.g. a goat, a sheep or a rabbit, following the usual and known immunization procedures, e.g. in the form of an emulsion in Freund's adjuvant, to originate anti-KlPk antiserum. This is recovered bleeding the animal according to usual techniques and is then absorbed, e.g. through incubation, with P" or pk antigenic determinants. Incubation is generally performed between room temperature and about 50"C, preferably at about 37"C, for a time varying from 1 to 5 days, preferably for about 3 days.Normal human plasma can be used instead of P" antigenic determinants since, as already said, normal human plasma contains P" antigenic determinants. Indicatively, for example, 0.1 ml of goat serum may be incubated at 37 C for 3 days with 324 ml of a PBS solution containing P" or pk antigenic determinants at a concentration of about 50 w/ml or, alternatively, with about 50 ml of normal human plasma. At the end of the incubation the material is centrifugated and the supernatant is used as such, or, possibly, after further dilution/purification/stabilization treatments, as the anti-K specific antibody.In accordance with the second above described method for preparing anti-K antibodies or antisera, P" or pk antigenic determinants are injected, e.g. in the form of emulsion in Freund's adjuvant, into an animal, e.g. a goat, a sheep or a rabbit, following conventional immunization procedure, to originate antigen or anti-Pk antiserum, which is recovered by bleeding the animal in an usual manner too.

Immunoglobulins are extracted from the antiserum; if desired, by known procedures.

The purification of the K and pk antigenic determinant mixture by means of antigen or anti-Pk antibodies to give the K antigenic determinants, may be carried out according to known methods. An useful technique may be, for example, the immunoaffinity, or bioabsorption, chromatography, which is based upon the immunological reaction between an antigen and an antibody bound to an inert support.

Thus, for example, a solution, e.g. a PBS solution, of the K and pk antigenic mixture is eluted through a resin, e.g. Sepharose 4B, carrying antigen or anti-Pk antibodies bound to it. The K antigenic determinants are so recovered in the unbound fraction.

Anti-K antiserum is then produced injecting the so obtained K antigenic determinants into an animal, e.g.

of the species indicated above, according to conventional procedures, for example those previously described in this specification.

The mixture of the antigenic determinants K and pk employed according to the above described processes for preparing anti-K antibodies is obtained by extraction from primary or metastatic adenocarcinoma tissues, e.g. from hepatic metastases, or from biological fluids of patients with adenocarcinoma.

Extraction may be carried out following known procedures and, if adenocarcinoma tissues are used, these require to be previously homogenized: generally homogenization is obtained with X Press (LKB).

Extraction may then be performed by any suitable solvent, preferably a glycoprotein solvent, which may be, for instance, perchloric acid; trichioroacetic acid; phosphotungstic acid; an alkali metal halide, e.g.

potassium chloride; phenol-ethanol mixtures; neutral, cationic or anionic detergents such as, for example, SDS, DOC, CPC, Triton X 100, Nonidet P40, and the like.

Preferably the extraction solvent is used in an equal volume as to the homogenate or biological fluid volume and it is, preferably, a concentrated acid, e.g. of a concentration from about 0.5N to about 2N: 2N perchloric acid is a particularly preferred extraction solvent. Any temperature below the room temperature is suitable for the extractive process even though a temperature, e.g. of about 4"C, is generally preferred. The extraction times usually vary from about ten minutes to about an hour.

After extraction, the precipitate is filtered off or centrifugated and the supernatant may be dialyzed and purified. Many known purification systems can be adopted, based upon different principles.

Thus, for example, on the basis of electric charge differences, ion exchange resins such as, e.g., DEAE Sephadex0, Sephacels or CM cellulose, or QAE Sephadex or SP Sephadex may be used for purification.

On the basis of molecular weight and hydrodinamic volume differences, chromatography techniques, such as, e.g., gel filtration and high performance liquid chromatography, may be foilowed.

A higher purification degree may be obtained on the basis of the differences in the electrophoretic migration and in the isoelectric point: thus electrophoretictechniques may be employed such as, for instance, preparative electrophoresis on inert support, e.g. Sephadex, polyacrylamide gel and the like, or plate or column isoelectrofocusing.

On the basis of its glycidic content, a further purification degree may be achieved, if desired, through the use of different lectines bound to resins, e.g., Sepharoses 4B.

The P" antigenic determinants to be used in the above described processes for producing anti-K antibodies, may be obtained from normal human plasma according to conventional extraction and purification procedures.

Thus, for example, normal human plasma may be extracted with, e.g., an equal volume of perchloric acid or with any other suitable solvent such as, for example, one of those indicated hereabove for the extraction of the WPk antigenic mixture from adenocarcinoma tissues; similar extraction temperature and times may be used too. The extraction product may be then dialyzed in a conventional manner and purified by known techniques, for example by immunoaffinity with antibodies specific to the P" determinants, e.g. by immunoaffinity chromatography against anti-K/Pk antibodies or against anti-Pk antibodies or against antigen anti bodies.The antigenic determinants P" are so bound to the antibodies and are then detached therefrom by known methods, e.g. by the use of thiocyanate or urea or propionic acid, according to conventional procedures [J.W. Eveleigh, D.E. Levy, J. of Solid Phase Biochemistry, 2,45, 1977].

The pk antigenic determinants which, if desired, may be involved too in the above processes for preparing anti-K antibodies may be, e.g., obtained from the mixture of the antigenic determinants K and pk. This mixture, extracted, e.g., from adencarcinoma tissues as previously described, may be, e.g., submitted to bioabsorption chromatography, by eluting, e.g., a solution thereof, e.g. a PBS solution, through a resin, e.g.

Sepharoses 4B, carrying antigen antibodies bound to it.

The pk antigenic determinants, as a consquence of their cross-reaction with the P" antigenic determinants, become linked to the resin carrying the anti-P" antibodies and are then detached therefrom in a conventional manner, e.g. by means of thiocyanate or urea according to conventional procedures, as reported above.

The contents or purification degree of the antigenic material, i.e. K, pk and P" antigenic determinants and K/Pk antigenic mixtures, are monitored, either radioimmunologically or immunochemically, on the basis of their K and pk or P" immunological activity, according to conventional methods. Thus, for example, a radioimmunological control of the K determinant contents of an antigenic material may be performed through measure of the inhibition produced by said material against the binding capacity of a RIA 1251-K anti-K system.Analogous control against a RIA 1251-Pk < anti.Pk system or, respectively, against a RIA 1251-P" o anti-P" system, allows to evaluate the pk or, respectively, the P" contents of an antigenic material.

The anti-P" antibodies and anti-Pk antibodies are obtained by the known immunization techniques, e.g. the same before described for obtaining anti-k/Pk antiserum and antibodies.

According to the invention, monoclonal anti-K, anti-Pk and anti-P" antibodies may be produced against a single K, pk or P" antigenic determinant following the known procedures generally used to prepare monoclonal antibodies, e.g. the known hybridoma technique as reported, e.g., by Kohler C. and Milstein C. in Nature 256,495-497, 1975. According to the invention, any known method for the determination of an antigen, for example any immunoassay method, e.g. radioimmunoassay (RIA), or enzymeimmunoassay (EIA), or immunofluorescence or immunoluminescence, or immunodiffusion, or immunoelectrophoresis method, may be employed to reveal or quantitate tha antigenic determinants K specific to adenocarcinomas or, if desired, the antigenic determinants pk associated with the K determinants on the adenocarcinomas.

The use of one or more of the antigenic determinants K, pk and KiPk antigenic mixture, either unlabelled or in their labelled form, and of the corresponding either unlabelled or labelled antibodies in anyone of the above said known methods allows to make early diagnosis of those human tumoral pathologies which are known as adenocarcinomas.

A labelled antigen, or antigenic determinant or antigenic mixture or antibody is, respectively, an antigen or antigenic determinant or antigenic mixture or antibody which has been tagged by means of any possible kind of tagging system: this may be, for instance. a radioisotope, an enzyme, or a fluorescent or a luminescent substance. For the tagging any known and usual procedure described, e.g., for labeiling antigens, may be followed.

To label, for instance, with 1251 or 1311 radioisotopes the Chloramine T or Lactoperoxidase (LPO) techniques [Nature 194,495, 1962 and, respectively, Bioch. Bioph. Acta 251,363, 1971], or modifications thereof may be followed; alternatively the Bolton and Hunter reagent [Biochem. J. 133, 529, 1973] may be used. To label, for instance, with an enzyme, the techniques reported in FEBS Letter 95,311, 1978 or in J. Histochem. Cytochem.

22,1084,1974, may be followed.

When a radioimmunoassay (RIA) method is used for the diagnosis of the adenocarcinomas, any known RIA techniques [see, e.g., Clin. Chem. 19:146, 1973] can be employed. Thus, for example, the antigenic determinants K specific to adenocarcinomas or the antigenic determinants pk associated with them on adenocarcinomas may be, for instance, quantitated in, e.g., a biological fluid or tissue extract by making them to compete with a known amount of radioisotope labelled, e.g. 1251 or 1311 radioiodinated, K or, respectively, pk antigenic determinants, or K/Pk antigenic mixture, for the reaction with a limited amount of anti-K or, respectively, anti-Pk antibodies.

The competition reaction may be carried out in a conventional way, e.g. by incubation at a temperature from about 4"C to about 40"C, preferably at room temperature, for a time varying approximately from 6 to 90 hours, preferably for about 15-16 hours.

The antigenic fraction bound to the antibodies is then separated from the unbound fraction, and the radioactivity is measured in the bound and1or unbound fraction.

The separation of the bound antigenic fraction from the unbound one may be carried out by known methods, e.g. by the use of antibodies specific to the immunoglobulins of the species wherein the first antibodies, i.e. anti-K or anti-Pk antibodies, have been originated.

The radioactivity in the bound and unbound fraction may be evaluated by means of a standard curve, traced e.g. with standard K or standard pk, or directly measuring the possible fall in the binding capacity of the radiolabelled K or Pk or K/Pk with the anti-K or anti-Pk antibodies, and so, for example, a decrease in radioacitivity measured on the bound fraction is proportional to the K or pk concentration of the sample.

When an enzymeimmunoassay method is employed for the diagnosis of adenocarcinomas, any known immunological method using an enzyme label may be employed, e.g. the methods described in J.

Immunology 109, 129, 1972.

Although many enzymes may be successfuliy used as label, particularly preferred enzymes are, for instance, peroxidase, p-gaiactosidase, alkaline phosphatase, and glucose oxidase.

Thus, for example, according to a possible enzymeimmunoassay procedure, the antigenic determinants K or pk of a sample from biological fluids or tissue extracts can be quantitated by a EIA sandwich-type procedure wherein the sample is made to react, e.g. by incubation, with anti-K or, respectively, anti-Pk antibodies which are either chemically bound or passively absorbed on a solid support, e.g. polystyrene beads.After eliminating the excess reagent by washings, enzyme labelled anti-K or, respectively, anti-Pk antibodies are added, such as, for example ss-galactosidase labelled antibodies obtained according to the procedure described in FEBS Letters 95,311-313, 1978. After further washings the appropriate enzymatic substrate is added, e.g. o-nitrophenyl-galactopyranoside if ss-galactosidase is used as enzyme label, and the developed colour is measured, its intensity being proportional to the concentration of the K or, respectively, the pk antigenic determinants in the sample.For the histochemical application any known immunohistochemical method [Human Pathology 12, 590-596, 1981] may be used to locate the presence of antigenic determinants K or pk on the histological preparates by means of anti-K or anti-Pk specific antibodies, according to the invention. The histological determination may be carried out on frozen or routinely fixed and embedded tissue sections according to conventional procedures.

Thus, for example, sections may be incubated at room temperature in an aqueous buffer with anti-K or, respectively, anti-Pk antibodies so that the anti-K or, respectively, anti-Pk antibodies will fix to the sites where the K or, respectively, pk antigenic determinants are present. The fixed antibodies are then evidentiated by subsequent reaction, e.g. incubation, with labelled antibodies specific to the immunoglobulins of the species wherein the anti-K or anti-Pk first antibodies have been originated.

The said labelled antibodies may be prepared according to known procedures using known labels such as, for instance, enzymes, e.g. peroxidase, B-galactosidase, alkaline phosphatase, glucose oxidase, enzyme-anti enzyme complexes, e.g. the PAP peroxidase-antiperoxidase complex; fluorescent molecules, e.g. fluorescein or rodamin; or Biotin-Avidin systems wherein Avidin is in its turn bound to an enzyme or to a fluorescent substance, e.g. of the kind mentioned hereabove.

The final microscopic analysis of the histological preparate will reveal the sites where the searched K or pk antigenic determinants are present: fluorescent sites will be observed when a fluorescent label is involved, while coloured sites will be observed when an enzyme label and a chromogenic substrate specific thereto are used in the determination.

A particularly useful system for the histochemical demonstration of K or pk antigenic determinants on tissue sections involves the ss-galactosidase enzyme as the label and an indolyl-galactoside, e.g.

5-bromo-4-chloro-indolyl-galactoside, as the enzymatic chromogenic substrate: the immuno-galactosidase technique described, e.g., in Histochemistry 76, 153-158, 1982, may be, e.g., followed for such determination.

As already said, however, besides the above discussed immunoassay procedures, any other immunological method may be employed, according to the invention, for demonstrating the presence of adenocarcinoma specific K antigenic determinants or associated pk antigenic determinants, in biological fluids or tissue extracts or sections.

As already said too, in addition to methods for the diagnosis of the human adenocarcinomas, the invention provides also reagent systems for carrying out the said methods.

Although different reagent systems are provided for different analytical methods, a reagent system for the demonstration of the K antigenic determinants contains always, as an essential component, an anti-K specific antibody, and a reagent system for the demonstration of the pk antigenic determinants contains, as an essential component, an anti-Pk antibody.Each of said reagent systems contains, additionally, other components which may be different from one reagent system to another depending on the analytical method the reagent system is intended for, and these additional components may be, for example, chosen from labelled K or pk antigenic determinants, labelled K/Pk antigenic mixtures, labelled anti-K or anti-Pk antibodies or labelled antibodies specific to the immunoglobulins of the species wherein the said anti-K or anti-Pk antibodies have been originated. Furthermore other components, either optional or not, may be present in the reagent systems provided by the invention such as, e.g., a bound-free separation system, controls, standards, buffers, stabilizing agents, antimicrobials, tensioactive substances, enzyme substrates and activators.

Thus, for example, for a radioimmunoassay method for the detection of the K or pk antigenic determinants in biological fluids or tissue extracts according to the RIA procedure hereabove described in this specification, an useful reagent system may be a kit essentially containing: 1) anti-K or, respectively, anti-Pk antibody; 2) radiolabelled K or, respectively, pk antigenic determinants or, in alternative, a radiolabelled K/Pk antigenic mixture; 3) controls; and 4) buffers.

The above kit may additionally contain a second antibody specific to the immunoglobulins of the species in which the anti-K or, respectively, anit-Pk antibody of the component 1) has been raised, and, optionally, tensioactive substances and/or stabilizing or antimicrobial agents.

The radiolabelled K or pk or K/Pk or the kit component 2) may be, e.g., K or Pk or K/Pk labelled with '251 or 1311 radioisotope.

The controls 3) involve at least one negative control and one positive control, and the buffers 4) may include any buffer having, e.g., a pH between about 6.5 and about 8, preferably about 7.2.

Tensioactive substances may be, for example, Tween 20 or 80 Nonidet P40, Triton X100, and the like.

Stabilizing agents may be, for example, bovine serum albumin, gelatin, and the like.

An antimicrobial agent may be, e.g., sodium azide.

Buffers may be, for example, phosphate buffers, possibly admixed with inorganic salts such as, e.g., sodium chloride.

In a particularly preferred feature a kit for the RIA detection of K or pk antigenic determinants contains: 1) an anti-K or anti-Pk antibody raised in goat, properly diluted in a mixture of - phosphate buffer having a pH between about 6.5 and about 8, preferably about 7.2, and a molar concentration between about 20 mM and about 150 mM, preferably about 70 mM; - sodium chloride at a molar concentration between about 20 mM and about 150 mM, preferably about 70 mM; - EDTA disodium salt at a molar concentration between aboout 25 mM and about 50 mM, preferably about 33 mM; - sodium azide at a molar concentration between about 15 mM and about 50 mM, preferably about 31 mM, and - goat normal serum at a volume to volume concentration between about 0.05% and about 0.5%, preferably about 0.1%;; 2) a reference buffer (to be used to determine the maximal binding capacity of the antibody radio-labelled antigen system) comprising: - phosphate buffer having pH between about 6.5 and about 8, preferably about 7.2, and a molar concentration between about 20 mM and about 150 mM, preferably about 70 mM; - sodium chloride at a molar concentration between about 20 mM and about 150 mM, preferably about 70 mM; - sodium azide at a molar concentration between about 15 mM and about 50 mM, preferably about 31 mM; and - bovine serum albumin at a weight to volume concentration between about 0.1% and about 1%, preferably about 0.4%; 3) a labelled tracer comprising;; '251 radioiodinated K or pk antigenic determinants or K/Pk antigenic mixture, diluted in the reference buffer 2) so as to have a concentration of 1.5 mg/ml with a specific activity of about 60 mCi/mg 4) a precipitating antiserum anti-goat-immunoglobulins, properly diluted in the reference buffer 2); 5) a negative control: human plasma negative to the test i.e. normal human plasma; and 6) a positive control: human plasma positive to the test i.e. human plasma from a patient with adenocarcinoma or normal human plasma to which a known amount of K or pk antigenic determinants or K/Pk antigenic mixture has been added.

For an enzymeimmunoassay method for the detection of the K or pk antigenic determinants in biological fluids or tissue extracts according to the EIA sandwich-type procedure before described in this specification, a reagent system may be used essentially containing, for instance: 1) anti-K or, respectively, anti-Pk antibodies bound to a solid phase; 2) enzyme conjugated anti-K or, respectively, anti-Pk antibodies; 3) an enzymatic substrate; 4) controls; and 5) buffers.

The above reagent system may additionally contains other components such as, for instance, a blocking agent of the enzymatic reaction and, furthermore, as already said, tensioactive substances, stabilizing agents, antimicrobials, enzyme activators, and the like.

The anti-K or anti-Pk antibodies of the component 1) may be, e.g., absorbed on polystyrene beads.

The enzyme conjugate component 2) may be, for example, anti-K or anti-Pk antibodies conjugated with the ss-galactosidase enzyme, according to known procedures as previously stated in this specification.

The enzymatic substrate component 3) may be, e.g., any chromogenic substrate which is specific to the enzyme of the component 2); if ss-galactosidase is used as enzyme a suitable chromogenic substrate may be, for instance, o-nitrophenylgalactopyranoside.

The controls 4) and buffers 5) may be as previously specified for the RIA kit.

The possible blocking agent of the enzymatic reaction may be, for instance when ss-galactosidase is used as enzyme, an alkali metal carbonate, e.g. sodium carbonate.

In a particularly preferred feature a kit for the above said ElA sandwich-type detection of the K or pk antigenic determinants comprises: 1) anti-K or, respectively, anti-Pk antibodies absorbed on polystyrene beads; 2) a diluent buffer containing:: - a phosphate buffer having pH between about 6.5 and about 8, preferably about 7.2, and a molar concentration between about 50 mM and about 150 mM, preferably about 70 mM; - sodium chloride at a molar concentration between about 50 mM and about 150 mM, preferably about 70 mM; - sodium azide at a molar concentration between about 15 mM and about 50 mM, preferbly about 31 mM; and - bovine albumin at a weight to volume concentration between about 0.5% and about 3%, preferably about 1%; 3) ss-galactosidase-conjugated anti-K or, respectively, anti-Pk antibodies, properly diluted in the buffer 2); 4) an enzymatic substrate containing: - o-nitro-phenylgalactopyranoside at a molar concentration between about 2 mM and about 4 mM, preferably about 2.3 mM; and - a phosphate buffer at a pH between about 6.5 and about 8, preferably about 7.2, and at a molar concentration between about 10 mM and about 30 mM, preferably about 15 mM; 5) a blocking agent of the enzymatic reaction containing sodium carbonate at a molar concentration between about 0.25 M and about 2 M, preferably about 1 M; 6) a negative control: human plasma negative to the test, i.e. normal human plasma; and 7) a positive control: human plasma positive to the test, i.e. human plasma from a patient with adenocarcinoma or normal human plasma to which a known amount of K or pk antigenic determinants or K/Pk antigenic mixture has been added.

For the histochemical assay of K or pk antigenic determinants on tissue sections according to the procedure previously described in this specification, an useful reagent system may be, e.g., a kit essentially containing: 1) anti-K or, respectively, anti-Pk antibodies; 2) enzyme labelled antibodies specific to the immunoglobulins of the species in which the anti-K or, respectively, anti-Pk antibodies of the component 1) have been raised; 3) an enzymatic substrate; and 4) buffers.

Other components may be included in the above kit, such as e.g., a blocking agent to eliminate possibly interfering aspecific interactions, a contrast colouring agent (for the nuclei) and, furthermore, as already said, tensioactive substances, stabilizing agents, antimicrobials, enzyme activators, and the like.

In the above reagent system the anti-K or anti-Pk antibodies may be, e.g., originated in goat and then the antibodies of the component 2) will be anti-goat-immunoglobulins antibodies and will be raised, e.g., in donkey. The enzyme labelled antibodies of the component 2) may be, e.g., p-galactosidase labelled antibodies obtained by known techniques as previously said.

The enzymatic substrate component 3) may be any substrate which is specific to the enzyme of the component 2) and which is able to give a coloured and insoluble product by the action of the enzyme: if ss-galactosidase is used as enzyme, an useful chromogenic substrate may be, for example, an indolylgalactoside, in particular 5-bromo-4-chloro-indolyl-galactoside.

The buffers may be, e.g., phosphate orTris buffers having a pH between about 6.5 and about 8, preferably about 7.3.

The blocking agent possibly required to eliminate interferences from aspecific interactions, may be, for example, normal donkey serum for the case that the component 2) antibodies are originated in donkey.

The contrast colouring agent may contain, e.g., carminic acid.

According to a particularly preferred feature a kitforthe histochemical demonstration of K or pk antigenic determinants on tissue sections contains: 1) a washing buffer comprising - Tris or phosphate buffer having a pH between about 6.5 and about 8, preferably about 7.3, and a molar concentration between about 5 mM and about 100 mM, preferably about 10 mM; and - sodium chloride at a molar concentration between about 50 mM and about 200 mM, preferably about 150 mM; 2) a blocking buffer comprising the same components of the washing buffer according to 1) above, and, in addition, normal donkey serum at a volume to volume concentration between about 1% and about 5%, preferably about 3%.

3) anti-K or, respectively, anti-Pk antibodies raised in goat, properly diluted in the buffer 1) above; 4) p-galactosidase conjugated anti-goat immunoglobulins antibodies, raised in donkey, properly diluted in the buffer 1) above; 5) an enzymatic substrate containing:: - a phosphate buffer having a pH between about 7 and about 8, preferably about 7.4, and a molar concentration between about 5 mM and about 50 mM, preferably about 10 mM; - sodium chloride art a molar concentration between about 5 mM and about 50 mM, preferably about 10 mM; - magnesium chloride at a molar concentration between about 0.1 mM and about 2 mM, preferably about 1 mM; - 5-bromo-4-chloro-indolylgalactoside at a molar concentration between about 0.5 mM and about 5 mM, preferably about 1 mM; - potassium ferrocyanide at a molar concentration between about 3 mM and about 10 mM, preferably about 6 mM; and - potassium ferrycyanide art a molar concentration between about 3 mM and about 10 mM, preferably about 6 mM; 6) a contrast colouring agent containing: - carminic acid at a weight to volume concentration between about 1 g/l and about 10 g/l, preferably about 2.5 g/l; and - aluminium potassium sulphate at a molar concentration between about 25 mM and about 150 mM, preferably about 50 mM.

In the preferred features described above for the reagent systems of the invention the reported concentrations indicate the concentrations per liter at which each component is present in the solution or mixture employed for carrying out the test.

The use of the reagent systems provided by the invention in the known analytical methods for the determination of the antigens, including liquid phase immunoassay procedures and solid phase immunoassay procedures and histochemical determinations, allows to improve in a relevant way the possibility to diagnose the human adenocarcinomas.

The possibility of detecting, according to the invention, only those antigenic determinants (K) which are specific to the adenocarcinomas without interference deriving from the antigenic determinants P" or, alternatively, those antigenic determinants (pk) which are associated with the K determinants on the adenocarcinomas, allows to achieve diagnostic results superior to those obtained with the known, commercially available kits for the CEA assay; with the methods and reagents of the invention indeed a highly reduced number of false positives is observed.

The results obtained from experimentation conducted with the methods and reagents of this invention on numerous cases of non-neoplastic pathologies, including many inflammatory-degenerative pathologies, and also of neoplastic pathologies, where characterized by percentages of false positives and, respectively, false negatives, lower than those observed in the cases when the known kits for CEA, employing anti-CEA antibidies, (e.g. the CEA Roche test and the Liso-phase CEA kit) are used.

When, in particular, comparative experimentation was conducted by analysis of histological preparates with the reagents of the invention and with commercially available kits, e.g. DAKO PAP KIT and IMMULOK HISTO SET, it was observed that, while the known kits give positive reaction also on some normal cells, with an error particularly high, e.g., in case of analysis of bone marrow tissues, the reagents of the invention reveal exclusively adenocarcinoma metastatic cells, with no false positive results at all, i.e. without any positive staining of the normal cells, e.g. ganulocytes, in distinct contrast with the compared commercially available kits.

The new, either polyclonal or monoclonal anti-K antibodies or, alternatively, anti-Pk antibodies provided by invention, as well as anti-K or anti-PK antibody tragments, e.g. Fab' or F(ab')2 fragments, may be used as tracers in, e.g., nuclear medicine and radioimmunochemistry. Thus, for example, the said antibodies or antibody fragments may be labelled with radioisotopes, e.g. 1311, and the so obtained radiolabelled tracers may be injected into the human body to visualize and localize possible adenocarcinoma cells or masses. The visualization of the tracer may be carried out by known methods through the use of particular instruments, for instance by -camera-scanning.

A further application of the new, either polyclonal or monoclonal anti-K antobidies, or anti-Pk antibodies, or fragments thereof, as provided by the invention, is for the therapy of the human adenocarcinomas. The possibility of using specific anti-K antibidies or respectively, anti-Pk antibodies which are able to recognize exclusively the adenocarcinoma cells without recognizing the normal cells, allows to realize a tumor chemotherapy which is specifically and exclusively directed to the tumor masses.

For this application the above said new antibodies or antibody fragments may be conjugated with anti-tumor drugs or other cytotoxic agents, and injected into the patient so that the drug or the cytotoxic agent is specifically conveyed by the antibody carrier only to the tumor cells or masses.

Anti-tumor drugs may be, for example, daunorubicin, doxorubicin, epirubicin and methotrexate. A cytotoxic agent may be, for example, ricin.

The conjugation of the anti-tumor drug or cytotoxic agent, e.g. one of those hereabove mentioned, with a polyclonal or monoclonal anti-K or anti-Pk antibody of the invention may be carried out following known procedures, for instance those described in J. Immun. Methods 59, 129-143 (1983). The conjugates between an anti-tumor drug or cytotoxic agent, e.g. one of those previously indicated, and a polyclonal or monoclonal anti-K or anti-Pk antibody, are a further object of the invention.

The present invention provides therefore also an improved method for a specific and focalized chemotherapy of the human adenocarcinomas, wherein the drug-effect is made to develop exclusively on the tumor cells and the normal cells are not affected at all by the drug-effect. In this specification the abbreviations EDTA, Tris, PBS, PEG, HPLC, lgG, mCi, mM, WN, VN, DEAE, SDS, DOC, CPC stand, respectively, for ethylendiamino-tetra-acetic acid, tris-(hydroxymethyl)-aminomethane, phosphate buffered saline, polyethyleneglycol, high performance liquid chromatography, immunoglobulin(s), milliCurie, millimolar, weight to volume, volume to volume, diethylaminoethyl cellulose, sodium dodecyl sulphate, sodium deoxycholate and cetyl pyridinium chloride.

The following examples illustrate but do not limit in any way the invention.

Example 1 Colon adenocarcinoma hepatic metastases (100 g) were frozen to -80 C for 24 hours and then homogenized under high pressure by X-press LKB instrument. The homogenate was taken up with 3 volumes of a 0.025 M aqueous solution of saccharose and then an equal volume of 2N He104 was added thereto. After centrifugation (10,000 x g x 20 minutes art 400) and dialysis of the supernatant against distilled water, precipitation was carried out with 3M KCI for 24 hours art 400 under stirring. The mixture was again centrifugated (100,000 x g x 1 hour at 4"C) and the supernatant then dialyzed against a pH 6.75 0.005 M phosphate buffer.

The extract was controlled by RIA on the basis of the produced inhibition of the binding capacity of a 1251-K anti-K or 1251-K/Pk anti-K RIA system and then chromatographed on a DEAE-cellulose column (1.6 x 5 cm) previously equilibrated with a pH 6.75 0.005M phosphate buffer. Elutions are successively performed with pH 6.75 0.025M phosphate buffer (40 ml), with pH 6.75 0.05M phosphate buffer (40 ml), and then with pH 6.75 0.1 M phosphate buffer (40 ml).Each eluted fraction was controlled by RIA as hereabove reported, then concentrated to 5 ml. the 0.025M and 0.05M fractions were further purified by gel filtration (4 ml/h) through a Sephadex G 200 column ( = 1.6 cm; h = 100 cm) buffered to pH 4.65 with an aqueous solution containing 0.05M NaH2PO4 and 0.9% NaCI. Two peaks were so eluted and each of them was again controlled by RIA, as indicated above, and also with a 1251-Pk anti-Pk system, in order to know the K and pk antigenic activity contents. Both peaks were confirmed to be a mixture of K and pk antigenic determinants with a K concentration higher in the first peak than in the second peak.

Example 2 Normal human plasma (51) was centrifugated (5,000 x g x 15 minutes art400) then the supernatant was extracted with an equal volume of 1.2N He104.

The extract was dialyzed against running water for 24 hours and then against bidistilled waterfor other 24 hours. After 10 to 20-folds concentration byAmicon cell and dialysis against pH 7.2 PBS, 0.5% Tween 80 was added to the extract (to avoid non specific interactions), and this was then chromatographed on a Sepharoses 4B bioabsorption column carrying anti-P" antibodies bound to the resin.

The fraction become linked to the resin during the elution process (P" antigenic determinants) was then detached from the resin itself by means of a 3M PBS solution of ammonium thiocyanate according to the procedure described in J. of Solid-Phase Biochemistry 2, 45, 1977.

The obtained product was dialyzed against distilled water and against pH 7.2 PBS, and then controlled by RIA through measure of the inhibition of the binding capacity of a '251-P" < anti-P" RIA system.

The contents was so confirmed to be of P" antigenic determinants at a concentration of 50 wimp, determined by Lowry method.

Example 3 A pH 7.2 PBS solution containing Tween 80 (0.5%) and the K/Pk antigenic mixture (1 mg/ml) obtained in example 1 (first eluted peak), was chromatographed on a Sepharoset 4B bioabsorption column binding anti-Pk antibodies.

The unbound fraction containing K antigenic determinants was concentrated to a small volume and controlled by RlAthrough measure of the inhibition of the binding capacity of a 1251-K < anti-K RIA system.

The unbound fraction was so confirmed to contain K antigenic determinants at a concentration of 0.1 mg/ml in the PBS-Tween mixture.

The bound fraction, i.e. the fraction which become linked to the column anti-Pk antibodies, was detached by elution with a 3M PBS solution of ammonium thiocyanate according to the procedure reported in example 2. The eluate was then dialyzed against bidistilled water and then against PBS, and its contents was controlled by RIA on the basis of the inhibition of the binding capacity of a 1251-Pk o anti.Pk RIA system. The eluted fraction was so confirmed to contain pk antigenic determinants at a concentration of 0.15 mg/ml.

The same above procedure was repeated using a Sepharosee 4B bioabsorption column binding antigen antibodies. During the elution of the K/Pk antigenic mixture, the pk antigenic determinants became linked by cross-reaction to the column anti-P" antibodies, and this was proved by the fact that pk antigenic determinants at a concentration of 0.15 mg/ml were recovered from the bound fraction. The pk contents control was again performed by RIA as reported above against a 1251-Pk e anti-Pk RIA system.

Example 4 A PBS solution of the K/Pk antigenic mixture obtained according to example 1, at a 1 mg/ml concentration, was emulsified with an equal volume of a complete Freund's adjuvant suspension. The obtained mixture was used to make 4 subcutaneous injections in 4 different sites in the back of a Tibetan goat. The injections were repeated three times at intervals of 15 days. After the third injection the immunological response was checked: the animal was bled and the serum title was controlled as follows. Serial dilutions of serum were incubated overnight at 37"C with 100 1 of a PBS solution containing 1.5 ng/ml of 1251 radioiodinated K antigenic determinants or, in an alternative procedure, of 1251 radioiodinated K/Pk antigenic mixture.

After incubation, 0.1 ml of a 1/10 PBS solution of anti-goat immunoglobulins raised in rabbit and 0.1 ml of a 1/1000 PBS solution of goat normal serum were added.

The mixture was then centrifugated (5,000 x g x 15' at 4 C), the supernatant was removed and the radioactivity was measured by gamma-counter on the precipitate. The title of the antiserum was calculated as the antibody dilution capable of binding 50% of the total labelled antigen. The obtained anti-K/Pk antiserum was found to have an average title of 1/50,000.

Following analogous procedure but using, instead of the K/Pk antigenic mixture, K antigenic determinants or pk antigenic determinants, obtained acccording to example 3, or P" antigenic determinants obtained according to example 2, anti-K antiserum, anti-Pk antiserum and anti-P" antiserum were obtained, respectively.

Example 5 The anti-K/Pk antiserum (1 ml) obtained in example 4, was treated with a 14% PEG 6,000 solution in pH 8.6 0.1M Veronal buffer (9 ml).

The obtained precipitate was taken up with the minimum amount of 0.02M phosphate buffer having pH 8, and purified on DEAE cellulose.

The proteins eluted in the starting buffer (5 ml) were precipitated again with 20% PEG 6,000 solution in pH 8.60.1 M Veronal buffer (45 ml). The precipitate was taken up with PBS and 0.5% Tween 80 (1 ml) and further purified on a Sepahadex 4B bioabsorption column carrying K/Pk antigens bound to it.

The anti-K/Pk immunoglobulins which remained bound to the column were then removed by elution with ammonium thiocyanate according to the procedure reported in example 2 to give anti-K/Pk antibodies.

By analogous procedure, starting from the anti-K, anti-Pk and anti-P" antisera obtained in example 4, anti-K, anti-Pk and anti-P" antibodies were obtained, respectively.

Example 6 The anti-K/Pk antiserum obtained according to the procedure of example 4 (0.1 ml) was incubated for 24 hours at 37"C with normal human plasma (8 ml) and the mixture was centrifugated (5000 x g x 15' at 4"C).

Each 0.1 ml of the supernatant were incubated again for 24 hours at 37"C with 4 ml of normal human plasma, and again centrifugated in the same conditions reported hereabove.

The supernatant was separated and its immunological activity was controlled on the basis of its capacity to bind at the same extent a 1251 radiolabelled K/Pk antigenic mixture both in presence and in absence of normal human plasma. The absence of cross-reaction with normal human plasma, i.e. with P" antigenic determinants, proved that the obtained antiserum was anti-K antiserum.

The obtained serum was brought to the proper dilutions and used for fhe immunological determinations of K antigenic determinants on human plasma or other biological fluids or tissue extracts.

Example 7 The anti-K/Pk antiserum obtained according to the procedure of example 4 (0.1 ml) was incubated for 24 hours at 37"C with a 50 y/ml PBS solution of P" antigenic determinants, obtained according to the procedure of example 2(1.8 ml). After incubation the mixture was centrifugated (5000 x g x 15' at 4"C).

Each 0.1 ml of the supernatant were in their turn re-incubated for 24 hours at 37"C with the 50 Fy/ml PBS solution of P" antigenic determinants (1.8 ml).

After centrifugation in the same hereabove said conditions, the supernatant was brought to the proper dilutions and used as anti-K antiserum for the immunohistochemical determinations.

The anti-K immunological activity of the antiserum was controlled on the basis of its capacity to reveal adenocarcinoma cells but not normal cells, on histological sections: for the test the peroxidase staining procedure was followed, as described by Sternberger L.A. in Immunocytochemisty - 2nd Edition - John Wiley and Sons - New York, 1979.

Example 8 BABBLE mice 8 weeks old were immunized by intraperitoneal injection of the emulsion obtained admixing K antigenic determinants, prepared according to the procedure of example 3 (100 cos), with an equal volume of complete Freund's adjuvant.

After 30 days, injections were repeated intraperitoneum, with lower amounts of K antigenic determinants.

Control of the immunological response was performed on the animals, as reported in example 4.

Using then animals positive to the control, somatic hybridization with murine myeloma cells was carried out according to the procedure described in Nature 256,495-497, 1975.

The specificity of the obtained products was evaluated by RIA test through measure of the binding capacity of a 1251-K antigen.

The hybrids which resulted positive to the test were then isolated and cloned.

Large amounts of anti-K monoclonal antibody were obtained by intraperitoneal injection into BALB/c mice of cells secreting the monoclonal anti-K antibody.

The growth of the tumor due to the injected cells displayed as the production of an ascitic liquid containing up to 20 mg/ml of monoclonal anti-K antibodies. Operating in analogous manner but using for the immunization pk or P" antigenic determinants, instead of K antigenic determinants, anti-Pk monoclonal antibodies and, respectively, anti-P" monoclonal antibodies were obtained.

Example 9 A solution of K antigenic determinants prepared according to the procedure of example 3 (10 > 9) in pH 7.2 PBS (20 FI) was treated for 5 minutes, in an ice bath, with a solution of Naa251 (1 mCi) in PBS (10 yl) and a solution (20 1) containing Chloramine-T in PBS at a concentration of 0.5 mg/ml.

Then 20 Ì of a solution containing sodium metabisulfite in PBS at a concentration of 1 mgiml, and 20,us of a solution containing potassium iodide in PBS at a concentration of 20 mg/ml were added to the mixture.

The reaction product was purified by HPLC in order to eliminate free 1251, and so '251 radioiodinated K antigenic determinants (1251-K) were quantitatively obtained.

By analogous procedure radioiodinated pk antigenic determinants (1251 pk) and radioiodinated K/Pk antigenic mixtures (1251-K/Pk) were prepared.

Example 10 Human serum samples from 335 hospitalized patients affected by non neoplastic diseases were tested by means of the anti-K antiserum obtained in example 6, using the reagents and the analytical procedure herebelow indicated.

Reagents (1) Reference buffer containing: - pH 7.2 phosphate buffer 70mM; -sodium chloride 70 mM; -sodium axide 31 mM; and - bovine albumin 0.4% WIV.

(2) Anti-K antiserum obtained in example 6, properly diluted 1:50,000 in: - pH 7.2 phosphate buffer 70 mM; -sodium chloride 70 mM; - EDTA disodium salt 33 mM; - sodium azide 31 mM; and - normal goat serum 0.1 VN.

(3) Radioiodinated label containing 1251-K/Pk antigenic mixture diluted in buffer (1) so as to have a concentration of 1.5 ng/ml and a specific activity of 60 mCi/mg.

(4) Second antiserum: antiserum precipitating goat immunoglobulins, originated in rabbit and properly diluted in buffer (1).

(5) Negative control: normal human plasma.

(6) Positive control: normal human plasma to which a known amount of K/Pk antigenic mixture has been added.

Analyticalprocedure "Zero" Negative Positive Sample control control Reference buffer (1) 0.5 ml - - Negative control (5) - 0.5 ml - Positive control (6) - - 0.5 mi Sample - - - 0.5 ml Anti-K antiserum (2) 0.2 ml 0.2 ml 0.2 ml 0.2 ml Mix, incubate5 hours at room temperature.

Radioiodinated label (3) 0.1 ml 0.1 ml 0.1 ml 0.1 ml Incubate overnight at room temperature Second antiserum (4) 0.2 ml 0.2 ml 0.2 ml 0.2 ml Incubate 1 hours at room temperature, centrifuge 30 minutes at 4,000 rpm, decant and measure radioactivity in the precipitate by gamma-counter.

Calculation: the P.l. positivity index was calculated evaluating the radioactivity of the samples, and, respectively, of the controls, with respect to the radioactivity of the "zero", according to the following expressions: activity of sample P.l. (sample) = 100 - x 100 activity of "zero" activity of control P.l. (control) 100 - x 100 activity of "zero" A P.l. index value higher than 15 was considered to be indicative of presence of adenocarcinoma, while a P.l. index value lower than 15 was considered to be indicative of absence of adenocarcinoma.

The above said 335 samples were also parallelly examined by using two commercially available kits for the detection of the human CEA, namely the CEA Roche Test, and the Liso-phase CEA Kit from Lepetit.

The obtained results are summarized in the following table: false positives number of cases pathologies Roche test Lepetit test our test 30 liver cirrhosis 4(13.3%) 3(10%) 2(6.6%) 17 pulmonary 4(23.5%) 4(23.5%) 2(11.7%) inflammatory diseases 18 obstructive 3(16.6%) 3(16.6%) 1(5.5%) broncopneumo pathies 270 other non 4(1.5%) 5(1.8%) 1(0.3%) neoplastic diseases 335 total 15(4.5%) 15(4.5%) 6 (1.8%) Example 17 Human serum samples from 84 patients affected by ascertained adenocarcinomas were analyzed by using the reagents and analytical procedure described in example 10 and, parallelly, by using the two commercial kits mentioned in example 10.

The results are summarized in the following table: false negatives number of cases pathologies Rochetest Lepetittest our test 60 gastrointestinal 5 (8.3%) 7 (11.6%) 2 (3.3%) adenocarcinomas 24 lung 2 (8.3%) 2 (8.3%) 0 (0%) adenocarcinomas 84 total 7 (8.3%) 9 (10.7%) 2.(2.4%) Example 12 The same human plasma samples of the examples 10 and 11 were also examined by using the reagents and the alternative analytical procedure reported below.

Reagents (1) Anti-K antibodies obtained according to the procedure described in the examples 5 and 6, absorbed on polystyrene beads (2) Diluent buffer containing: - pH 7.2 phosphate buffer 70 mM; - sodium chloride 70 mM; -sodium azide 31 mM; and -bovine albumin 1% (WN).

(3) Enzymatic conjugate containing anti-K antibodies as those of the reagent (1) above, conjugated to -galactosidase (according to the procedure described in FEBS Letters 95, 311, 1978) and properly diluted in buffer (2).

(4) Enzymatic substrate containing: - pH 7.0 phosphate buffer 15 mM; and - o-nitrophenylgalactoside 2.3 mM.

(5) Blocking agent: 1 M sodium carbonate.

(6) Negative control: normal human plasma.

(7) Positive control: normal human plasma to which a known amount of K/Pk antigenic mixture has been added.

Analytical procedure "Zero" Negative Positive Sample control control Diluent buffer (2) 0.2 ml 0.1 ml 0.1 ml 0.1 ml Negative control )6) - 0.1 ml - Positive control (7) - - 0.1 ml Sample - - - 0.1 ml Anti-K antibodies (1) 1 bead 1 bead 1 bead 1 bead Incubate under shaking for 5 hours at room temperature.

Discard the liquid phase.

Wash 2 times with 2 ml of saline solution.

Enzymatic conjugate (3) 0.2 ml 0.2 ml 0.2 ml 0.2 ml Incubate under shaking for 2 hours at room temperature.

Discard the liquid phase.

Wash 2 times with 2 ml of saline solution.

Enzymatic substrate (4) 1 ml 1 ml 1 ml 1 ml Incubate for 1 hour at 37 C.

Blocking agent (5) 1 ml 1 ml 1 ml 1 ml Measure absorbance at 420 nm by spectrophotometer.

Calculation The positivity index P.l. was calculated with respect to the optical density (O.D.) of the "zero" according to the expression P.I. = O.D. of sample P.1. = O.D. of "zero" A positivity index value higher than 3 was considered to be indication of presence of adenocarcinoma, while a positivity index value lower than 3 was considered to be indication of absence of adenocarcinoma.

Similar results as those reported in the examples 10 and 11 were observed.

Example 13 Formalin fixed and paraffin embedded tissue sections from human colon adenocarcinoma, human mammalian adenocarcinoma and from bone marrow of patients with breast adenocarcinoma were examined using the reagents and the analytical procedure indicated below: Reagents (1) Washing buffer containing: - pH 7.3 phosphate buffer 10 mM; and - sodium chloride 150 mM.

(2) Blocking buffer containing: - pH 7.3 phosphate buffer 10 mM; - sodium chloride 150 mM; and - normal donkey serum 3% (VN).

(3) Enzymatic conjugate containing anti-goat immunoglobulins antibodies raised in donkey, labelled with -galactosidase (according to the method described in FEBS Letters 95,311, 1978) and properly diluted in buffer (1).

(4) Enzymatic substrate containing: - pH 7.4 phosphate buffer 10 mM; - sodium chloride 10 mM; - magnesium chloride 1 mM; - 5-bromo-4-chloro-indolyl galactoside 1 mM; - potassium ferrocyanide 6 mM; and - potassium ferricianyde 6 mM.

(5) Contrast colouring agent containing: - aluminium potassium sulphate; and - carminic acid.

(6) Anti-K antibodies raised in goat, obtained according to the procedure of example 7, and properly diluted in buffer (1) above.

Analytical procedure Sections were dewaxed in xylol, in the 100% to 50% ethyl alcohol series, and then in the washing buffer (1).

Any reagent was added in such an amount to have the whole section covered.

Blocking buffer (2).

Incubate 20 minutes at 37 C in moist chamber.

Eliminate the excess reagent by shaking.

Add anti-K antibodies (6).

Incubate 2 hours at 37 C in moist chamber.

Add enzymatic conjugate (3).

Incubate 30 minutes at 37"C in moist chamber.

Wash the section with buffer (1) for 5 minutes.

Add enzymatic substrate (4).

Incubate 30 minutes at 3700.

Eliminate the excess reagent by washing.

Add the contrast colouring agent (5).

After 1 minute wash the section in running water and observe by light microscope.

In alternative the above analytical procedure was followed performing incubations overnight at room temperature in moist chamber, except for the incubation with the enzymatic substrate (4), which was carried out for 1 hour at room temperature in moist chamber.

Always as an alternative in the above procedure, after the final washing in running water and before examination at the microscope, the sections were first clarified by passages through the washing buffer (1), the 50% to 100% ethyl alcohol series, and xylol, and then mounted in Eukitt.

The microscope analysis of the sections revealed an intensely blue staining exclusively localized to the cytoplasm and the membrane of the tumoral cells without any positive staining reaction in the cells of the adjacent normal tissue such as, e.g., granulocytes.

These results were found to be in distinct contrast with those obtained analysing the same preparates by the use of the commercially available kits for the histochemical detection of the CEA complex through anti-CEA antibodies, namely the DAKO PAP KIT and the IMMUNOLOK HISTOSET KIT.

The said commercial reagents showed, in fact, very lower specificity in that they were found to stain not only the tumor cells but also, to a various degree, the normal tissue cells, with an error particularly high in the case of analysis of bone marrow tissues.

The same above procedure was followed for analysis of frozen tissue sections and analogous results were obtained.

Example 14 The doxorubicin was conjugated to the anti-K antibodies by a covalent linking method.

A solution of doxorubicin (40 mg/ml) in pH 7.2 0.015M phosphate buffered saline was mixed with a slight molar excess of 0.1 M NalO4 and incubated for 1 hour at room temperature in the dark.

At the end of the incubation 1 M glycerol was added up to a 0.05M final concentration.

The solution of oxidized doxorubicin was mixed with 1 ml of anti-K antibodies solution (20 mg/ml) in pH 9.5 0.15M potassium carbonate buffer and incubated for 1 hour at room temperature.

At the end of the incubation NaBH4 was added up to a final concentration of 0.3 mg/ml and the reaction was allowed to proceed for 2 hours at 37 C.

At the end of the reaction, the mixture was chromatographed on a gel-filtration column (15 x 1.5 cm) of Bio-gels p 100 equilibrated in PBS.

The fractions in the excluded volume contained the antibody bound-drug free from the unconjugated drug.

Claims (49)

1. A method for use in diagnosing a human adenocarcinoma by determining adenocarcinoma-related antigenic material in a sample taken from a human patient, which method comprises determining material in the sample having (a') exclusively those antigenic determinants K specific to adenocarcinomas or (b') those antigenic determinants pk associated with the K determinants on the adenocarcinomas using, respectively, (a) anti-K antibody or (b) anti-Pk antibody.

2. Method according to claim 1, wherein the sample taken from a human patient is a sample from a biological fluid or tissue extract or section.

3. Method according to claim 1 or 2, wherein the determination of the K, or respectively, pk, antigenic determinants is carried out qualitatively, quantitatively or semi-quantitatively.

4. Method according to any one of the preceding claims, wherein monoclonal anti-K antibody or monoclonal anti-Pk antibody is used.

5. Anti-K antibodies specific to the K antigenic determinants.

6. Process for the preparation of the anti-K antibodies of claim 5, said process comprising absorbing antibodies produced against a mixture of K and pk antigenic determinants with P" or pk antigenic determinants, or pk antigenic determinants, so as to saturate the pk immunological binding sites on the said antibodies.

7. Process for the preparation of the anti-K antibodies of claim 5, said process comprising: 1) immunizing animals with P" or pk antigenic determinants to produce anti-P" or anti-Pk antibodies; 2) purifying a mixture of antigenic determinants K and pk by means of the anti-P" or anti-Pk antibodies produced according to 1), so obtaining K antigenic determinants; and 3) immunizing animals with K antigenic determinants obtained according to 2).

8. Anti-Pk antibodies specific to the pk antigenic determinants.

9. Process for the preparation of the anti-Pk antibodies of claim 8, said process comprising immunizing animals with pk antigenic determinants.

10. Anti-P" antibodies specific to the P" antigenic determinants.

11. Process for the preparation of the anti-P" antibodies of claim 10, said process comprising immunizing animals with P" antigenic determinants.

12. Antigenic determinants K specific to the human adenocarcinomas.

13. Process for the preparation of the K antigenic determinants of claim 12, said process comprising purifying a mixture of antigenic determinants K and pk by means of anti-P" or anti-Pk antibodies.

14. Antigenic determinants pk, said antigenic determinants pk being those antigenic determinants associated with the K antigenic determinants on the human adenocarcinomas.

15. Process for the preparation of the pk antigenic determinants of claim 14, said process comprising purifying a mixture of antigenic determinants K and pk by means of anti-P" antibodies.

16. Antigenic determinants P", said antigenic determinants P" being those antigenic determinants present in normal plasma or serum and cross-reacting with the pk antigenic determinants.

17. Processforthe preparation of the P" antigenicdeterminants of claim 16, said process comprising extraction from normal plasma or serum and subsequent purification by immunoaffinity with antibodies specific thereto.

18. Monoclonal anti-K antibodies specific to single K antigenic determinants.

19. Monoclonal anti-Pk antibodies specific to single pk antigenic determinants.

20. Monoclonal anti-P" antibodies specific to single P" antigenic determinants.

21. Processes for the preparation of the monoclonal antibodies of the claims 18, 19 or 20, wherein the said monoclonal antibodies are obtained by the hybridoma technique.

22. A reagent system for use in a method for the diagnosis of human adenocarcinomas according to any one of claims 1 to 4, comprising as an essential component, anti-K antibodies specific to the antigenic determinants K.

23. A reagent system according to claim 22 comprising monoclonal anti-K antibodies.

24. A reagent system for use in a method for the diagnosis of human adenocarcinomas according to any one of claims 1 to 4, comprising, as an essential component, anti-Pk antibodies specific to the antigenic determinants pk.

25. A reagent system according to claim 24 comprising monoclonal anti-Pk antibodies.

26. A reagent system for use in a radioimmunoassay method for the diagnosis of human adenocarcinomas, according to any one of claims 1 to 4, essentially containing: 1) anti-K or, respectively, anti-Pk antibody; 2) radiolabelled K or, respectively, pk antigenic determinants or, in the alternative, a radiolabelled K/Pk antigenic mixture; 3) controls; and 4) buffers.

27. A reagent system according to claim 26 containing, as further component, a second antibody specific to the immunoglobulins of the species in which the anti-K or, respectively, anti-Pk antibody of the component 1) has been raised.

28. A reagent system according to claims 26 or 27 wherein the radiolabelled component 2) is 1251-K or pk or 125i-K/Pk.

29. A reagent system for use in an enzyme-immunoassay method for the diagnosis of the human adenocarcinomas according to any one of claims 1 to 4, essentially containing: 1) anti-K or, respectively, anti-Pk antibodies bound to a solid phase; 2) enzyme conjugated anti-K or, respectively, anti-Pk antibodies; 3) an enzymatic substrate; 4) controls; and 5) buffers.

30. A reagent system according to claim 29 wherein the enzyme of the component 2) is ss-galactosidase and the enzymatic substrate component 3) comprises o-nitro-phenyl galactopyranoside.

31. A reagent system for use in an immunohistochemical method for the diagnosis of the human adenocarcinomas, according to any one of claims 1 to 4, essentially containing: 1) anti-K, or, respectively, anti-Pk antibodies; 2) enzyme labelled antibodies specific to the immunoglobulins of the species in which the anti-K or, respectively, anti-Pk antibodies of the component 1) have been raised; 3) an enzymatic substrate; and 4) buffers.

32. A reagent system according to claim 31 wherein the enzyme of the component 2) is t3-galactosidase and the enzymatic substrate component 3) comprises 5-bromo-4-chloro-indolyl-galactoside.

33. Labelled K, pk and P" antigenic determinants or labelled anti-K, anti-Pk and anti-P" polyclonal or monoclonal antibodies.

34. Labelled antigenic determinants or antibodies according to claim 33 wherein the said antigenic determinants and antibodies are radiolabelled.

35. Labelled antigenic determinants or antibodies according to claim 33 wherein the said antigenic determinants and antibodies are enzyme-labelled.

36. Polyclonal or monoclonal anti-K or anti-Pk antibodies for use as carriers of anti-tumor drugs or cytotoxic agents in the therapy of the human adenocarcinomas.

37. Conjugates between a polyclonal or monoclonal anti-K or anti-Pk antibody and an anti-tumor drug or a cytotoxic agent.

38. Conjugates according to claim 37 wherein the anti-tumor drug is chosen from daunorubicin, doxorubicin epirubicin and methotrexate.

39. Conjugates according to claim 37 wherein the cytotoxic agent is ricin.

40. A process for the preparation of antigenic determinants P", said antigenic determinants P" being the antigenic determinants present in normal plasma or serum, said process being substantially as hereinbefore described in Example 2.

41. A process for the preparation of antigenic determinants K specific to a human adencarcinoma, said process being substantially as hereinbefore described in Example 3.

42. A process for the preparation of antigenic determinants pk, said antigenic determinants pk being the antigenic determinants associated with the K antigenic determinants on a human adenocarcinoma, said process being substantially as hereinbefore described in Example 3.

43. A process for the preparation of anti-K-antibodies specific to K antigenic determinants, said process being substantially as hereinbefore described in any one of Examples 1 to 7.

44. A process for the preparation of anti-Pk antibodies specific to pk antigenic determinants, said process being substantially as hereinbefore described in Example 4 or 5.

45. A process for the preparation of anti-P" antibodies specific to P" antigenic determinants, said process being substantially as hereinbefore described in Example 4 or 5.

46. A process for the preparation of a monoclonal anti-K antibody, anti-Pk antibody or anti-P" antibody specific to a single K antigenic determinant, pk antigenic determinant or P" antigenic determinant, respectively, said process being substantially as hereinbefore described in Example 8.

47. A process for the preparation of radioiabelled antigenic determinants K, pk or P", said antigenic determinants being the determinants specific to a human adenocarcinoma, associated with K antigenic determinants on a human adencarcinoma or present in normal plasma or serum, respectively, said process being substantially as hereinbefore described in Example 9.

48. A method for use in the diagnosis of a human adenocarcinoma, said method being substantially as hereinbefore described in any one of Examples 10 to 13.

49. A conjugate of a polyclonal or monoclonal anti-K or anti-Pk antibody and an anti-tumor drug or a cytotoxic agent, said conjugate being substantially as hereinbefore described in Example 14.