DE3337465A1 - Antigenic determinants which are related to adenocarcinoma, specific antibodies for them, process for preparing them, and reagent systems containing them - Google Patents

Abstract

A method suitable for the diagnosis of human adenocarcinomas is described. In the method, antigenic material which is related to adenocarcinomas is determined in a sample which is taken from a human patient. The material in the sample is determined by using (a) anti-K antibodies or (b) anti-P<k> antibodies, using (a') exclusively those antigenic determinants K which are specific for adenocarcinomas, or (b') those antigenic determinants P<k> which with the K determinants are specific for adenocarcinomas.

Description

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"'"''' Description

The invention relates to new antigenic determinants which are related to adenocarcinomas, antibodies specific therefor and methods for diagnosing human adenocarcinoma, those based on the demonstration of antigenic determinants in biological fluids, in tissue extracts or sections and in vivo of tumor cells or masses based.

The invention further relates to the use more specifically Antibodies in the therapy of adenocarcinoma for carriage or the delivery of anti-tumor drugs or other cytotoxic agents to the tumor cells and masses. It is known that in cases of tumor pathology it is possible from the tumor masses themselves or from the biological fluids Isolate substances associated with the pathologies are. Many of these substances have antigenic properties, more common in animals than in humans. Some of them, which are generally referred to as "tumor markers", are oncofetal antigens. The presence of an antigen that is said to be primarily related to adenocarcinoma and particularly associated with their metastases, Gold et al:; ',. J. / expt. ..Med. 121: 439-462 (1965). This Antigen is known as k; a: r: 2i: naembryonic antigen (CEA) and can be detected both in biological fluids and in tissue extracts or sections by various known methods such as those described in the U.S. Patents 3,663,684 and 3,697,638, in Ann. of Clin. and Lab. Science, j4, 5, 357, 1974 in Clin. Chem. 1-9, 10, 1973, Cancer 34: 1504-1509, 1974, in Clin. Res. 19: 143, 1971 in Proc. Natl. Acad. ' may be. USA 64: 161-167, 1969, in Cancer 37: 62-81, 1976, in Scand. J. Immunol., Vol. 7, Suppl. 6, 1978, in Cancer 45: 1243-1247, 1980 and J. Natl. Cancer Inst. 57, 1, 1976.

However, the numerous studies in this field including, for example, the above references, have one resulted in very low specificity of CEA. It wasn't

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shown only in the case of tumoral pathologies, but also in the presence of various, i.e. non-tumor, pathologies, such as many degenerative inflammatory ones Lawsuits, and even in health donors. Furthermore, biochemical studies (Urba R., Alpert E. Isselbacher K.J., Proc. Nat. Acad. May be. USA 72, 4602-4606 (1975)) Differences between CEAs obtained from different laboratories have been shown, with the consequence that different ones clinical results obtained when the same Processes are used with materials of different origins.

It has now been found that it is possible to au :? human adcnocarcinoma metastases as well as from all human adenocarcinomas, for example gastric, mammary and thyroid adenocarcinomas, to isolate antigenic determinants (K) which are specific for adenocarcinomas, and ^{to isolate antigenic determinants (P u} ) which are associated with the K- Determinants associated with the adenocarcinoma. While the P ^<; antigenic determinants show a very strong cross-reaction with the antigenic determinants (P ⁿ ) present in the plasma or serum of normal donors, the K-antigenic determinants do not show any cross-reactivity with the P -antigenic determinants. The mixture of the antigenic determinants K and P and possibly some other unknown antigens was previously known as CEA.

Using the above-mentioned antigenic determinants K

k η
and antigenic determinants P and P are used in accordance with the invention

k η

specific antibodies anti-K, anti-P and anti-P produced.

The use of antigenic determinants K and P and the antibodies anti-K and anti-P, which are specific to them, contribute the known immunological methods, such as radioimmunoassay, enzyme immunoassay, immunofluorescence, Immunoluminescence and many others, exclusively allow such antigenic determinants K, which are specific for adenocarcinomas

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are specific, to be detected and quantified, without interference caused by antigenic determinants P ⁿ of normal plasma or serum, or, if desired, those antigenic determinants P that are associated with the K determinants on the adenocarcinomas. The above determination can be carried out either qualitatively or quantitatively or semiquantitatively.

The invention relates to an improved method for diagnosing human adenocarcinomas by determining antigens Material which is related to adenocarcinomas is either in a sample taken from a human patient, for example in vitro on biological fluids or tissue extracts or sections or in vivo on the tumor cells or masses, which is characterized in that

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using specific anti-K or anti-P antibodies, exclusively those antigenic determinants K which are specific for adenocarcinomas are ^{determined without the P n} antigenic determinants of normal cells being detected, or those antigenic determinants P which are associated with the K determinants on the adenocarcinomas. This will reduce false positives.

The term "antigenic determinants K" means antigenic determinants which, as already stated, are specific for adenocarcinomas and which do not cross-react with the P ⁿ antigenic determinants of normal cells and which are only recognized by antibodies which are specific for them i.e. anti-K antibodies. This definition includes any CEA-antigenic mixtures in which the antigenic determinants other than the K-determinants have been completely or substantially removed.

The term "antigenic determinants P" means such antigenic Determinants which are present on the adenocarcinoma cells in "association" with, i.e. together with, the antigenic determinants K are present that are specific for the adenocarcinoma,

the P antigenic determinants, as already indicated, ^{showing a cross-reaction with the P n} antigenic determinants of normal cells. This definition includes anywhere.] Before ¹ CIiA antigenic mixtures in which the antigenic determinants, with the exception of the P -determinants, have been completely or substantially removed.

The P antigenic determinants are, as previously indicated, antigenic determinants found in normal plasma or serum are present and cross-react with the P antigenic determinants.

An anti-K antibody is either an antibody that can be obtained by immunizing animals with the K antigens mentioned above Determinants, or any anti-CEA antibody (i.e. an antibody produced when animals are exposed to CEA immunized) modified in such a way that the antibody sites excluding the anti-K sites, therein wholly or essentially by antigenic determinants, the are specific for them, are saturated.

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Similarly, an anti-P antibody is either an antibody obtained by treating animals with the above P antigens Determinants immunized, or any anti-CEA antibody modified in such a way that the antibody sites 'off-' taken the anti-P sites, therein wholly or essentially by antigenic determinants specific for them sin ^ i, have been saturated.

In the present application, the term "antibody" means both immunoglobulins and immunoglobulin fragments and it can also include the corresponding antisorum.

Unless otherwise indicated, the term antibody means a polyclonal antibody. When a monoclonal Antibody is meant, this is indicated.

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The present invention relates to two alternative methods for the production of anti-K antibodies for K antigens Determinants are specific. .

According to one method, anti-K antibodies are obtained by absorption of antibodies produced against

k η

a mixture of K and P antigenic determinants with P -

k k

or P -antigenic determinants, so that the P -immunological binding sites on the antibodies are saturated. ,. ' ., .: -

According to the other method, anti-K antibodies are obtained by

η k

1) ■ Immunization of animals with P or P antigenic

n k

terminal with the formation of anti-P or anti-P antibodies,

2) Purification of a mixture of antigenic determinants

k η k

K and P. means the anti-P or anti-P antibodies, produced according to 1), with the formation of K antigens Determinants, and

3) Immunization of animals with K-antigenic determinants obtained according to 2).

The invention also relates to the anti-K-specific Antibodies obtained by the two alternative methods above, including K-antigenic determinants, the were obtained according to step 2) of the second alternative process described above, and also the ·

k η k

P and P antigenic determinants and the anti-P and anti-P -Antibodies that are specific to this and that appear in the above procedures.

The invention also includes marked forms, i. radiolabeled, enzyme-labeled or fluorescence-labeled form

k η men of the aforementioned K, P and P antigenic determinants

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k η

and the corresponding anti-K, anti-P, and anti-P antibodies.

According to the first manufacturing method described above of anti-K antibodies, the mixture of yeast determinants becomes K and P are injected into an animal such as a goat, sheep or rabbit, and then the usual ones follow and known immunization methods, for example in the form

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an emulsion in Freund's adjuvant to anti-K / P antiserum to create. This is obtained by bleeding the animal using methods known per se and then adsorbed, both

n k

for example by incubation with P or P antigenic determinants. The incubation is generally carried out at a temperature between room temperature and about 50 ^° C., preferably about 37 ^° C., for a time which varies from 1 to 5 days, preferably for about 3 days. Normal human plasma can be used in place of P ⁿ antigenic determinants since, as already stated, normal human plasma contains P antigenic determinants. For example, adding 0.1 ml goat serum at 37 ⁰ C for 3 days with 324 ml PBS solution containing

η k
Contains P or P antigenic determinants at a concentration of about 50 AVmI, or alternatively incubate with about 50 ml normal human plasma. Towards the end of the incubation the material is centrifuged and the supernatant used as such or possibly after further dilution / purification / stabilization treatments as anti-K-specific antibodies. According to the second method described above for the production of anti-K antibodies or antisera, P ⁿ or P antigenic determinants, for example in the form of the emulsion in Freund's adjuvant, are injected into an animal, for example a goat, a sheep or a rabbit , and this is followed by the well-known immunization procedure,

η k

whereby anti-P or anti-P antiserum is formed, which is obtained by bleeding the animals in a manner known per se.

Immunoglobulins may be used according to known methods Procedure extracted from the antiserum.

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The purification of K and P antigenic determinants with-

n k

by means of anti-P or anti-P antibodies with the formation of K antigens Determinants can be carried out according to methods known per se. A suitable method is, for example the immunoaffinity or bioabsorption, chromatography, which-is based on the immunological reaction between an antigen and an antibody bound to an inert carrier.

For example, a solution, for example a PBS solution,

of the K- "and P-antigenic mixture by a resin, for example Sepharose 4B, which is tied to it anti-P - or antiquity

P antibodies are eluted. The K antigenic determinants are thus obtained in the unbound fraction.

Anti-K antiserum is then according to methods known per se, as previously described, formed by applying the K-antigenic determinants thus obtained to an animal, for example of the species indicated above.

The mixture of antigenic determinants K and P, the corresponding the method described above for the production of anti-K antibodies is used by extraction of primary or metastatic adenocarcinoma tissues, for example from hepatic metastases, or from biological fluids isolated from patients with adenocarcinoma.

The extraction can be carried out according to methods known per se and, if adenocarcinoma tissues are used, this requires homogenization beforehand. In general, the homogenization is obtained with an X press (LKB).

The extraction can then be carried out with any suitable solvent, preferably a glycoprotein solvent, which, for example, perchloric acid, trichloroacetic acid, Phosphotungstic acid, an alkali metal halide, for example potassium chloride, phenol-ethanol mixtures, new

neutral cationic or anionic detergents such as SDS, DOC, CPC, Triton X 100, Nonidct P. _Q , and the like.

The extraction solvent is preferred in the same volume, based on the homogenate or the biological fluid volume, is used and is preferably a concentrated acid, for example at a concentration of about 0.5N to about 2N to be used. 2N-perchloric acid is a particularly preferred

10, th extraction solvent. Any temperature below room temperature is suitable for the extraction process, although a temperature of, for example, about 4 ^° C. is generally preferred. Extraction times can vary from about 10 minutes to about 1 hour.

After the extraction, the precipitate is filtered off or centrifuged and the supernatant can then be dialyzed and purified will. Many known cleaning systems can be used, all of which are based on different principles.

For example, one can use the electrical charge differences, ion exchange resins such as DEAE Sephadex, Sephacel or CM-Cellulose, or QAE Sephadex or SP Sephadex, use for cleaning.

Due to the molecular weight and the hydrodynamic volume differences can chromatography methods such as Gel filtration and high performance liquid chromatography, be used.

A higher degree of purification can be achieved due to the differences in electrophoretic migration and isoelectric migration Reach point. Thus, electrophoretic methods can be used as such, for example preparative Electrophoresis on an inert support, for example Sephadex, polyacrylamide gel. and the like, or a plate or Column isoelectrofocusing.

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On the basis of the glycidin (glyciidic) content, a further degree of purification can optionally be obtained by using different lectins that are bound to the resins, for example Sepharose 4B. ' _: ..

The P antigenic determinants used in the methods described above for the production of anti-K antibodies can be produced from normal human plasma by extraction and purification processes known per se will.

For example, normal human plasma can be used with, for example, an equal volume of perchloric acid or any other suitable solvent, for example any of those indicated above for the extraction of K / P antigenic mixtures from adenocarcinoma tissues. Similarly, the extraction temperature and times can also be used. The extraction product can then be dialyzed in a manner known _{per se and purified by methods known per se f,} for example, by immunoaffinity with antibodies that are specific to P -determinants, for example by immunoaffinity chromatography against anti-

k k

K / P antibodies or against anti-P antibodies or against anti-P antibodies. The antigenic determinants P ⁿ are thus bound to the antibodies and they are derived therefrom by methods known per se, for example through the use of thiocyanate or urea or propionic acid, in accordance with the usual regulations (JW Eveleigh, DE Levy, J. of Solid Phase Biochemistry, 2, 45, 1977).

The P antigenic determinants, which may also be used in the above methods for the production of anti-K antibodies can occur, for example, can be obtained from the mixture of the antigenic determinants K and P.

This mixture, for example from Adenokarζinomgeweben, such as previously described, extracted, can bioabsorption chromatography eluting with, for example, a solution of

for example a PBS solution, by a resin, for example Sepharose 4B, which
wears, be subjected.

wise Sepharose 4B, which anti-P ⁿ antibody bound to it

The P -antigenic determinants as a consequence of their cross-reaction with P -antigenic determinants are transferred to the resin, which the anti-P antibody carries, bound and are then in a manner known per se, for example with thiocyanate or Urea, replaced by methods known per se, as mentioned above.

The contents or the degree of purification of the antigenic material,

k η
for example K, P and P antigenic determinants and

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K / P antigen mixtures, either with a radioimmunoassay or immunochemically on the basis of their K-

k η

or P- or P -immunological activity according to methods known per se. For example, a radioimmunological Control of the K determinant levels of an antigenic material be carried out by the measure of inhibition that this material has against a binding capacity

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from RIA JK <-> Anti-K-System results. Analog controls

125 k k

compared to an RIA JP <- > Anti-P system or or compared to an RIA JP ⁿ <-> Anti-P ⁿ system allows the assessment

k η

or or the calculation of the P or or P content of the antigenic Materials.

η k

The anti-P antibodies and anti-P antibodies are then used known immunization methods, for example the one previously used for the production of anti-K / P antiserum and antibodies method described.

According to the invention, monoclonal anti-K, anti-P and anti-P ⁿ antibodies to a single K, P or P ⁿ antigenic determinant, followed by the general methods as used for the production of monoclonal antibodies, for example according to the known Ilybridoma method, as described by Kohler C. and Mil-tein C. in Nature 25f>,

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^ 9 ^ .ß ^. ^. ^^^^. ^ & ^ I ^: A ^ & b '& n, - hexgB . According to the present invention, any known method for the determination of an antigen, for example any immunoassay method, for example a raäioximmunoassayverfahren (RIA) or an enzyme immunoassay method can be used. (EIA) or an immunofluorescence or an immunoluminescence or an immunodiffusion or an immunoelectrophoresis method can be used to detect the antigenic determinants K that are specific for adenocarcinomas or to determine quantitatively, or optionally the 10'antigenic determinants P that are associated with the K determinants associated with adenocarcinoma.

The use of one or more antigenic determinants

k 'k

K, P. 'And the K / P antigenic mixture, either unlabelled or in their labeled form, and of the corresponding either unlabeled or labeled antibodies to either The above mentioned method allows the early diagnosis of such human tumoral pathologies as adenocarcinomas are known.

A labeled antigen or an antigenic determinant or an antigenic mixture or an antibody is an antigen or an antigenic determinant or antigenic mixture or antibody produced by some type of labeling system has been marked; this can, for example, be a radioisotope, an enzyme or a fluorescent or luminescent agent, be. For the labeling, any known and previously described method for labeling antigens can be used use.

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125 131 For marking, for example, J or J radioisotopes can use the chloramine-T or lactoperoxidase (LPO) method (Nature 194, 195, 1962 and Bioch. Bioph. Acta 251, 363, 1971) or their modifications can be used will. Alternatively, the Bolton and Hunter reagent can be used (Biochem. J. 133, 529, 1973) '.

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For the labeling of, for example, with an enzyme, the methods described in FEBS Letter 95, 311, 1978 or in J. Histochem. Cytochem. 22, 1084, 1974, are used will.

When a radioimmunoassay (RIA) procedure is used for the diagnosis of Adenocarcinoma used can be any known one Use RIA methods (see e.g. Clin. Chem. 19: 146, 1973). For example, one can use the antigenic determinants K specific for adenocarcinoma or the antigenic determinants Quantitatively determine P associated with them in the adenocarcinoma, for example in a biological Fluid or a tissue extract by mixing them with a

1 25 known amount of radioisotope-labeled, for example J-

131 k

or J-radioionated, K or or P -antigenic determinants, a K / P -antigenic mixture for the reaction with a certain amount of anti-K- or anti-P -antibodies to compete. The competitive reaction can be carried out in a customary manner, for example by incubation at a temperature of from about 4 to about 40 ^° C., preferably at room temperature, for a time which varies from about 6 to 90 hours, preferably from about 15 to 16 hours will. :

The antigenic fraction bound to the antibodies is then separated from the unbound fraction and the radioactivity is determined in the bound and / or unbound fraction.

The bound antigenic fraction can be separated from the unbound fraction by methods known per se, for example using antibodies which are specific for immunoglobulins of the species in which the first antibodies, ie anti-IC or anti-P antibodies, are derived , be performed.
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The radioactivity in the bound and the unbound Fraction can be determined using a standard curve that begins with standard K

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1 or standard P labeled, or you can directly measure the possible decrease in the binding capacity of radiolabeled K or P or K / P ^k with the anti-K or anti-P antibodies, and so for example the decrease in the radioactivity measured in the bound fraction, proportional to the K or P concentration of the sample.

When using an enzyme immunoassay method for the diagnosis of adenocarcinoma is used, any known immunological method using an enzyme label can be used will, for example, use the methods described in J. Immunology 109, 129, 1972.

Although many enzymes are suitably used as a label particularly preferred enzymes are, for example, peroxidase, β-galactosidase, alkali phosphatase and glucose oxidase.

For example, according to a possible enzyme immunoassay method ahren the antigenic determinants K or P of a sample from biological fluids or tissue extracts determine quantitatively using an EIA sandwich method, in which the sample is, for example, incubated with anti-K-

or reacts accordingly with anti-P antibodies, which are either chemically bound or passively attached to a solid carrier, for example polystyrene beads, are absorbed. After eliminating the excess reagent by washing enzyme-labeled anti-K or anti-P antibodies added, such as antibodies labeled with ß-galactosidase, obtained according to the method as described in FEBS Letters 95, 311 to 313, 1978. After further Washing, the appropriate enzymatic substrate, e.g. o-nitrophenylgalactopyranoside, is added if β-galactosidase is used as an enzyme label and the developed color is determined. Their intensity is proportional the concentration of the K or P antigenic determinants

th in the sample. For histochemical application any known immunohistochemical methods (Human Pathology 12, 590-596, 1981) can be used to determine the presence of antigenic determinants K or p 'on the histological To determine preparations by means of anti-K or anti-P -specific antibodies according to the invention. The histological Determination can be based on frozen or routinely fixed and embedded tissue sections according to known ones Procedure.

For example, you can have sections at room temperature in an aqueous buffer with anti-K or anti-P antibodies inhibit, so that the anti-K or anti-P antibodies are fixed at the points where the K or P antigens Determinants are present. The fixed antibodies are then released by subsequent reactions, for example incubation, with labeled antibodies for the immunoglobulins of the species that make up the anti-K or anti-P first antibodies originate, are specific, detected.

The labeled antibodies can be produced by methods known per se using known labels such as, for example Enzymes such as peroxidase, ß-galactosidase, Alkali phosphatase, glucose oxidase, enzyme-antienzyme complexes, for example the PAP-peroxidase-antiperoxidase complex, fluorescent molecules, such as fluorescein or rodamine, or biotin-avidin systems, in which the avidin in turn bound to an enzyme, or a fluorescent substance, for example of the kind mentioned above will.

The final microscopic analysis of the histological specimens shows the places where the K or P antigens are sought Determinants are present. Fluorescent spots will be observed if a fluorescent label is present is, whereas colored spots are observed when an enzyme label and a chromogenic specific for it Substrate to be used in the determination.

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As previously indicated, in addition to the method for diagnosing human adenocarcinomas, the invention also relates Reagent systems for performing these procedures. Although different reagent systems for different analytical Procedures provided contain the reagent system for demonstration of the K antigenic determinants an anti-K-specific antibody and the reagent system for the demonstration

k '
tion of the P anti-gene determinants contains an anti-P antibody as an essential component. Each of these reagent systems additionally contains other components which differ from one reagent system to the other depending on the analytical method for which the reagent system is intended. These additional components can, for example, consist of

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can be selected from marked K or P antigenic determinants, labeled K / P antigen mixtures, labeled anti-K or anti-P antibodies or labeled antibodies containing. are specific for the immunoglobulins of the species from which the anti-K or anti-P antibodies are derived.

As already indicated, in addition to the radioimmune methods mentioned above any other immunological method according to the invention for the detection of the presence of adenocarcinoma specific K-antigenic determinants or associated P -antigenic determinants in biological fluids or Tissue extracts or sections are used.

A particularly useful system for histochemical demonstration of K or P antigenic determinants on tissue 'sections relates to the ß-galactosidase enzyme as a marker and an indolyl galactoside, for example 5-bromo-4-chloro-indolyl galactoside, as an enzymatic chromogenic substrate. The immunogalactosidase process, which is described for example in Histochemistry 76, 153-158, 1982, can be used as such for such a provision may be used.

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Furthermore, other components can optionally be included in the Be present reagent system, which is provided by the present invention, such as a binding-free separation system, comparisons, standards, buffers, stabilizers, antimicrobial agents, tensioactive Mibtcl, Enzyme substrates and activators.

For example, for a radioimmunoassay method for the detection of K or P antigenic determinants in biological Fluids or tissue extracts according to the RIA procedure described above a useful reagent system a kit or be a product which essentially contains:

1) Anti-K or anti-P antibodies, 15

2) radiolabeled K or P antigenic determinants or alternatively a radiolabeled K / P antigenic mixture,

3) Compare and
4) buffer.

The above kit can also contain a second antibody, that for the immunoglobulins of the species in which the anti-K or anti-P antibodies of component 1) are bred was, is specific, and it can optionally surfactant substances and / or stabilizers or antimicrobial Funds included.

k k The radiolabeled K or P or K / P of the kit component

k k 2) can be, for example, K or P or K / P labeled J or J isotopes.

The comparisons 3) include at least one negative comparison and one positive comparison and the buffers 4) can any buffers, for example with a pH of between about 6.5 and about 8, preferably about 7.2.

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Tensioactive substances can be, for example, Tween 20 or 80, Nonidet P ₄₀ / Triton X100 and similar substances.

Stabilizers can be, for example, bovine serum albumin,
Gelatin and "the like."

An example of an antimicrobial agent is sodium azide.
The buffers can be, for example, phosphate buffers, optionally mixed with inorganic salts, such as, for example, sodium chloride.

According to a particularly preferred embodiment contains a

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Kit for the RIA detection of K or P antigenic determinants
1) Anti-K or anti-P antibody found in a goat

was cultured, appropriately diluted in a mixture of phosphate buffer with 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 about 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 normal goat serum with a volume concentration between about 0.05% and
about 0.5%, preferably about 0.1% _f

2) a comparison buffer, which is used to determine the maximum binding capacity of the antibody frradio-labeled
Antigen system is used, which contains: a
Phosphate buffer with a pH between about 6.5 and about 8, preferably about 7.2, and a molar concentration

Tration 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, preferred

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?;, bc preferably about 0.4%,

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3) a labeled tracer that contains: J-radioiodine-

k k

Nated K or P antigenic determinants or K / P antigenic mixture, diluted in the comparison buffer 2), so that a concentration of 1.5 mg / ml with a specific activity of 60 mCi / mg is obtained,

4) Precipitation Antiserum Anti-Goat Immunoglobulins, appropriately diluted in the comparison buffer 2),

5) 'a negative control, versus human plasma negative the test, i.e. normal human plasma, and

6) a positive control, human plasma positive versus the test, i.e. human plasma from a patient with adenocarcinoma or normal human plasma, to the a known amount of K or P antigenic determinants or K / P antigenic mixture was added.

For an enzyme immunoassay method for the detection of K or P antigenic determinants in biological fluids or tissue extracts according to the EIA sandwich method previously described a reagent system can be used which essentially contains, for example:

1) Anti-K or anti-P antibodies, bound to a solid phase,

2) enzyme-conjugated anti-K or anti-P antibodies, 35

3) an enzymatic substrate,

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4) Compare and
5} buffer.

The above reagent system can additionally contain other components, such as a blocking agent for the enzymatic Reaction and further, ^ as already stated, tensioactive substances, stabilizers, antimicrobial agents, enzyme activators and the like included.

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The anti-K or anti-P ^V antibodies of component 1) can, for example, be absorbed on polystyrene beads.

The enzyme conjugate component 2) can, for example, anti-K-

k ■ '

or anti-P antibodies linked to the ß-galactosidase enzyme are conjugated according to methods known per se, as indicated above in this application.,

The enzymatic substrate component 3) can, for example, be any chromogenic substrate which is suitable for the enzyme component 2) is specific. If ß-galactosidase is used as the enzyme, it is a suitable chromogenic substrate for example o-nitrophenylgalactopyranoside.

As before for the RIA kit specified, can be selected.

The possible blocking agent for the enzymatic reaction can, for example, when ß-galactosidase is used as the enzyme will be an alkali metal carbonate such as sodium carbonate.

In a particularly preferred embodiment, the kit contains for the above-mentioned EIA sandwich detection of the K or P antigens Determinants

1) Anti-K or anti-P antibodies, absorbed on polystyrene beads,

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2) a dilution buffer that contains:

a phosphate buffer with a 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, preferably about 31 mM, and bovine albumin at a weight-to-volume concentration between about 0.5 and about 3%, preferably about 1%,

k 3) ß-galactosidase conjugated anti-K or anti-P -

Antibodies appropriately diluted in a 'buffer 2),

4) an enzymatic substrate which contains: 20

o-Nitrophenylgalactopyranoside at a molar concentration between about 2 mM and about 4 mM, preferably about 2.3 mM, and

a phosphate buffer with 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 (stopping agent) for the enzymatic reaction, which sodium carbonate at a molar concentration between about 0.2 5 M and about 2 M, preferably about 1 M,

6) a negative comparison: human plasma that is negative to the test, i.e. normal human Plasma, and

7) a positive comparison: human plasma that is positive to the test, i.e. human plasma from a patient with adenocarcinoma or normal human

■ '· k

Plasma to which a known amount of K or P antigenic determinants or K / P antigenic mixture was added.

For the histochemical assay of K or P antigenic determinants on tissue sections according to the method previously described in the present application, a useful The reagent system, for example a kit, essentially contains:

1) anti-K or anti-P antibodies,

2) enzyme-labeled antibodies that are directed against the immunoglobulins of the species in which the anti-K or or Anti-P antibodies of component 1) have been raised, are specific,

3) an enzymatic substrate and

4) buffer.

Other components that may be present in the above kit are, for example, a blocking agent to possibly prevent to eliminate disruptive specific interactions, a contrast dye (for example for the nuclei) and furthermore, as already stated, surfactant substances, stabilizers, antimicrobial agents, enzyme activators and similar.

In the above reagent system, the anti-K or anti-P 'antibodies be bred in a goat or goat, for example, and then the antibodies of component 2) Anti-goat immunoglobulin antibodies and they are for example bred in donkeys. The enzyme-labeled antibodies of the

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Component 2) can, for example, be labeled with ß-galactosidase Be antibodies which have been obtained by methods known per se, as indicated above.

The enzymatic substrate component 3) can be any substrate which is specific for the enzyme of component 2) and which is colored by the influence of the enzyme and gives insoluble product. When ß-galactosidase as an enzyme is used, useful chromogenic substrate for example an indolylgalactoside, especially 5-bromo-4-chloroindolylgalactoside, be.

The buffers can, for example, be phosphate or tris puff with a pH of about 6.5 to about 8, preferably about 7.3.

The blocking agents that may be required to eliminate the interference from aspecific interactions can be, for example, normal donkey serum in the event that component 2) antibodies are present in donkeys were bred.

The contrast dye can contain carminic acid, for example.
25th

According to a particularly preferred embodiment:; florin nines kits for the histochemical detection of K or P antigens This includes determinants in tissue sections:

1) a washing buffer that contains tris or phosphate buffer with a pH between about 6.5 and about 8, preferably about 7.3, and at a molar concentration between about 5 mM and about 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 that has the same components as the washing buffer contains according to 1) above and plus normal donkey serum at a volume-to-volume concentration contains between about 1 and about 5%, preferably about 3%,

if

3) Anti-K or anti-P antibodies, which are in a

Goat have been bred, suitably diluted in the buffer 1) above,
10

4) ß-galactosidase conjugated anti-goat immunoglobulin antibodies, which have been bred in a donkey, appropriately diluted in the above buffer 1),

5) an enzymatic substrate that contains:

a phosphate buffer with a pH between about 7 and about 8, preferably about 7.4, and a molar concentration between about 5 and about 50 mM, preferred about 10 mM,

0 sodium chloride at a molar concentration between about 5 and about 50 mM, preferably about 10 mM, Magnesium chloride at a molar concentration between about 0.1 and about 2 mM, preferably about 1 mM, 5-Bromo-4-chloroindolylgalactoside at a molar concentration between about 0.5 and about 5 mM, preferably about 1 mM,

Potassium ferrocyanide at a molar concentration between about 3 and about 10 mM, preferably about 6 mM, and potassium ferrycyanide at a molar concentration between 0 see about 3 and about 10 mM, preferably about 6 mM,

6) a contrast dye which contains:

Carminic acid at a weight-to-volume concentration between about 1 and about 10 g / l, preferably about 2.5 g / l, and

Aluminum potassium sulfate at a molar concentration between about 25 and about 150 mM, preferably about 50 mM.

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In the preferred embodiments described above for the reagent systems according to the invention mean those indicated Concentrations are the concentrations per 1 in which the component is in the solution or mixture that is used to carry out of the attempt is used.

The use of a reagent system according to the invention in a known analytical method for the detection of Antigens include liquid phase and solid phase immunoassay methods, immunoassay methods, and histochemical evidence and through these reagent systems the diagnosis of human adenocarcinomas is significantly improved.

The possibility, according to the invention, of only such antigenic determinants (K) that are specific for the adenocarcinoma, detect without interference caused by antigenic determinants P or, alternatively, antigenic determinants (P), which are associated with the K determinants on the adenocarcinomas are derived, enables diagnostic results that are better than those that can be obtained with those known commercially available kits for the CEA assay. Indeed, the methods and reagents of the present invention reduce the number of false positives.

The results obtained in experiments carried out with the methods and reagents according to the invention are numerous cases of non-neoplastic pathologies, including numerous inflammatory degenerative ones Pathologies, and also neoplastic pathologies. They are characterized by the fact that the percent ^ - false positives and correspondingly lower than one false negatives observed in cases where known kits for CEA using anti-CEA antibodies (for example the CEA-Roche-Test and the Lisophasen-CEA-Kit).

If, in particular, comparative experiments in the analysis of histological preparations with the reagents according to the invention and the commercially available kits, for example Dako Pap Kit and Immulok Histo Set, are carried out, it is observed that, while the known kits, positive reactions also in normal cells with a particularly high level Yield errors, ie in the case of the analysis of bone marrow tissues, the reagents according to the invention exclusively indicate adenocarcinomas, metastatic cells, without false positive results at all, ie without any positive discoloration of normal cells, for example granulocytes, in marked contrast to those commercially available comparable kits. The new either polyclonal or monoclonal anti-K antibodies or, alternatively, anti-P ^ antibodies provided by the invention, as well as any anti-K or anti-P antibody fragments, for example Fab ¹ - or F (ab ') ₂ "fragments can be used as tracers, for example in nuclear medicine and in radioimmunochemistry. For example, the antibodies or the antibody fragments can be used

131

elements are labeled with radioisotopes, for example J, and the radiolabeled tracers obtained in this way can be used in Injected into the human body to remove possible adenocarcinoma cells or to visualize and localize masses. The visualization (or visualization) of the tracer can according to methods known per se using special instruments, for example by ^ camera scanning (scanning) , take place.

0 Another application of the new either polyclonal or

v monoclonal anti-K antibodies or anti-P antibodies or their fragments, which are provided according to the invention is for the therapy of human adenocarcinomas. The possibility of using specific anti-K antibodies or anti-P antibodies, all of which are exclusive to the adenocarcinoma cells are recognized - without them being recognized by. normal cells are recognized, allows the realization

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tumor chemotherapy, which specifically and exclusively on '^; the tumor mass is targeted.

For this application, the above novel antibodies or antibody fragments can be combined with anti-tumor drugs or others cytotoxic agents are conjugated and used in the patient be injected so that the drug or the cytotoxic Agent is specifically transported by the antibody carrier only to the tumor cells or masses. '10

Anti-tumor agents can, for example, daunorubicin, doxorubicin, Be epirubicin and methotrexate.

A cytotoxic agent can be, for example, ricin.

Conjugation of antitumor drugs or cytotoxic Means, for example of one of the aforementioned, with a polyclonal or monoclonal according to the invention Anti-K or anti-P antibodies can be prepared by methods known per se, for example those as described in J. Immun. Methods 59, 129-143 (1983). The conjugates between an anti-tumor drug or a cytotoxic agent such as one as previously noted and an anti-K or anti-P "polyclonal or monoclonal antibody are also the subject of the present invention.

The invention further relates to an improved method for a specific and focalized chemotherapy of human Adenocarcinomas in which the drug action is developed exclusively in the tumor cells and in which the normal Cells are also not affected by the drug action. In the present application, the abbreviations mean EDTA, Tris, PBS, PEG, HPLC, IgG, mCi, mM, W / V, V / V, DEAE, SDS, DOC, CPC ethylenediaminotetraacetic acid, tris (hydroxymethyl) aminomethane, phosphate-buffered saline, polyethylene glycol, high-performance liquid chromatography,

Immunoglobulin (s), millicuries, millimolar, weight-to-volume, Volume-to-volume, diethylaminoethyl cellulose, sodium dodecyl sulfate, Sodium deoxycholate and cetyl pyridinium chloride.

The following examples illustrate the invention without representing it restrict.

example 1

^1% 0 Hepatic colon Adenokarzinommetastasen (100 g) frozen h at -8O ⁰ C during 24 and then under high pressure with an X-press LKB instrument homogenized. The homogenate is taken up in 3 volumes of a 0.025 M aqueous solution of sucrose and then the same volume of 2N HClO. admitted. After centrifugation (10,000 g 20 min at 4 ^° C.) and dialysis of the supernatant against distilled water, precipitation is carried out with 3M KCl for 24 h at 4 ^° C. with stirring. The mixture is centrifuged again (100,000 × g × 1 h at 4 ^° C.) and then the supernatant is dialyzed against a 0.005 M phosphate buffer at pH 6.75.

The extract is determined by means of RIA on the basis of the successful inhibition of the binding capacity of J-K <-> Antique-

5 or ¹²⁵ JK / P ^k £ -> anti-K-RIA system and then checked on a DEAE cellulose column (1.6 χ 5 cm) which was previously equilibrated with pH 6.75 0.005M phosphate buffer. The eluents are carried out successively 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 0.1M phosphate buffer (40 ml). Each eluted fraction is checked according to the RIA described above and then concentrated to 5 ml. The 0.025M and 0.05M fractions are further buffered to a pH of 4.65 by gel filtration (4 ml / h) over Sephadex G 200 column (0 = 1.6 cm _{; h = 100 cm)} with an aqueous solution containing 0.05M NaII ₂ PO ₄ and 0.9% NaCl, cleaned. So two peaks are eluted and each of them becomes new

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by means of RIA, as previously indicated, and also with a

125 k k

J-P <r-> Anti-P system controls to keep the levels at

k
K and P antigenic activity to be determined- It was confirmed

It indicates that both peaks are a mixture of K and P antigens Determinants are where the K concentration is in the first Peak is higher than the second peak.

Example 2

Normal human plasma (5 l) is centrifuged (5000 χ g χ 15 min at 4 ⁰ C) and then the supernatant with an equal volume of 1.2N HClO. extracted.

The extracts are dialyzed against running water for 24 hours and then against double-distilled water for a further 24 hours. After 10 to 20 times the concentration by an Amicon cell and dialysis against pH 7.2 PBS, 0.5% Tween 80 is added to the extract (to avoid specific intermediate reactions) and then on a Sepharose-4B -Bioabsorption column carrying anti-P ⁿ antibodies bound to the resin, chromatographiort.

The fraction that is attached to the resin during the> elution process (P antigenic determinants) is then bound by the resin itself using a 3M PBS solution of ammonium thioeyanate according to the method as described in J. of Solid-Phase Biochemistry 2, 45, 1977 is solved.

The product obtained is dialyzed against distilled water and against pH 7.2 PBS and then checked or analyzed ^{by means of RIA through the inhibition of the binding capacity of a 125} JP ¹¹ 4r ^ anti-P ^{n -RIA system.}

It was found that the levels of P ⁿ antigenic determinants are at a concentration x of 50 µg / ml as determined by the Lowry method.

Example3

A pH 7.2 PBS solution containing Tween 80 (0.5%) and the K / P ^k antigenic mixture (1 mg / ml) obtained according to Example 1 (first eluted peak) is applied to a Sepharose 4B bioabsorbent column that binds anti-P antibody, chromatographed.

The unbound fraction, which contains K-antigenic determinants, is concentrated to a small volume and measured by RIA by measuring the inhibition of the binding capacity of a
lates.

1 25
of a JK <-? Anti-K-RIA systems analyzed or controlled

It was thus confirmed that the unbound fraction was K antigens Contains determinants at a concentration of 0.1 mg / ml in the PBS-Tween mixture.

The bound fraction, i.e. the fraction bound to the column anti-P antibodies, is eluted detached with a 3M PBS solution of ammonium thiocyanate according to the method described in Example 2. The eluate is then dialyzed against double distilled water and then against PBS and its content is determined by means of the RIA on the Basis of the inhibition of the binding capacity of a

¹²⁵ JP ^k <- ^ Anti-P ^k -RIA system controlled. It was confirmed that the eluted fraction contained P antigenic determinants at a concentration of 0.15 mg / ml.

The above procedure is repeated using a Sepharose 4B bioabsorbent column that ^{binds anti-P n} antibodies.

During the elution of the K / P antigenic mixture, the P antigenic determinants are cross-reacting to the column anti-P -Antibodies bound and this is evidenced by the fact that P -antigenic determinants at a concentration of 0.15 mg / ml can be isolated from the bound fraction

T,

the. The P content is checked again by means of the RIA,

125 k k

as explained before, against a J-P <~> Anti-P-RIA system

checked.
Example4

A PBS solution of the K / P antigenic mixture, obtained according to Example 1, with a concentration of 1 mg / ml is emulsified in a mild equal volume of complete Freund's adjuvant suspension. The resulting mixture is used to make 4 subcutaneous injections at 4 different sites in the back of a Tibetan goat. The injections are repeated three times at 15 day intervals. After the third injection, the immunological response is checked. The animal is bled and the serum titer is checked as follows. Serial dilutions of the serum are overnight at 37 ⁰ C with 100 μΐ PBS solution,

125
which 1.5 ng / ml J-radioiodinated K-antigenic determinants

1 25 th or, in an alternative method, J-radioiodinated K / P antigenic mixture is incubated.

After incubation, 0.1 ml of a 1/10 PBS solution of anti-goat immunoglobulins produced in a rabbit and 0.1 ml of a 1/1000 PBS solution of normal goat serum are added. The mixture is then centrifuged (5000 χ g χ 15 min at 4 ⁰ C), the supernatant is removed and the radioactivity is determined with a gamma counter on the test precipitate. The title of the antiserum is calculated as the antibody dilution that can bind 50% of the total labeled antigen. The anti-K / P antiserum obtained has an average titer of 1/50000.

Analogous procedures were used, but instead of the

k k

K / P antigenic mixture, K antigenic determinants or P antigenic determinants, obtained according to Example 3, or P antigens Determinants obtained according to Example 2 are used; anti-

k 'η

K antiserum, anti-P antiserum and anti-P antiserum who

received accordingly. COPY

. .-. ^^^ r ^ - '· - Example 5

The anti-K / P antiserum (1 ml), obtained according to Example 4, is 0.1M veronal buffer with 14% PEG 6000 solution in pH 8.6 (9 ml) treated.

The precipitate obtained is taken up with the small amount of 0.02M phosphate buffer with pH 8 and purified on DEAE cellulose.
• 1 0 '

The proteins: eluted in the starting buffer (5 ml) are again with 20 PEG 6000 solution in pH 8.6 0, "IM veronal buffer (45 ml) precipitated. The precipitate is taken up with PBS and 0.5-5 Tween 80 (1 ml) and further on one Sephadex 4B bioabsorbent column with K / P antigens bound to it.

The anti-K / P immunoglobulins that remain bound to the column, are then eluted with ammonium thiocyanate according to the procedure described in Example 2 is removed, giving anti-K / P antibodies.

According to analogous procedures using anti-K-, An (i-

k η

P and anti-P antisera as starting material, get gel η

measured example 4, become anti-K, anti-P and anti-P antibodies obtain.

Example6

The anti-K / P antiserum, obtained according to the method of Example 4 (0.1 ml), is ^{incubated for 24 h at 37 °} C. with normal human plasma (8 ml) and then the mixture is centrifuged (5000 χ g 15 min at 4 ⁰ C). In each case 0.1 ml of the supernatant are ^{incubated again for 24 hours at 37 °} C. with 4 ml of normal human plasma and then centrifuged again under the same conditions as stated above.

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The supernatant was separated and its immunological activity is based on its capacity, in the same

125 k

Extent of binding a J-radiolabeled K / P antigone mixture both in the presence and in the absence of normal human plasma was examined. The absence of αΐηιτ cross-reaction with normal human plasma, ie with P ⁿ antigenic determinants, shows that the antisemite obtained! Anti-K antiserum is.

tO The serum obtained is reduced to the appropriate dilutions diluted and for immunological determinants of K-antigenic determinants in human plasma or other biological Fluids or tissue extracts are used.

Example7

The anti-K / P antiserum, obtained according to the method of Example 4 (0.1 ml), is obtained for 24 h at 37 ° C. with 50 J '/ ml PBS solution of P ⁿ antigenic determinants obtained according to the Procedure of Example 2, (1.8 ml) incubated. After the incubation, the mixture is centrifuged (5000 χ g 15 min at 4 ^° C.).

Depending 0.1 are reincubated succession during 2-1 h at 37 ⁰ C · 50) '/ ml PBS solution of P ⁿ i -ant gcnon Doterminahten (1.8 ml) ml of the supernatant.

After centrifugation under the aforementioned conditions, the supernatant is diluted to the appropriate dilutions and used as an anti-K antiserum for immunohistochromic determinations.

The anti-K immunological activity of the antiserum will be histological based on its capacity to display adenocarcinoma cells, not to display normal cells Sections determined. For the 'process, the peroxidase discoloration process, win from Sternbergor L.A.

in Immunocytocheinistry, 2nd Edition, John Wiley and Sons, New York, 1979, used.

Example 8 5

BALB / c mice, 8 weeks old, are injected with intraperitoneal Injection of the emulsion obtained by using K-antigenic determinants prepared according to the method of Example 3, (100 μg), with the same volume complete Freund's see adjuvants mixed, immunized.

After 30 days, injections are repeated intraperitoneum with smaller amounts of K-antigenic determinants. A Comparison of the immunological response is carried out with the animals as indicated in Example 4.

Using animal-positive results compared to the control, the somatic hybridization with mice or Rat myeloma cells according to the method described in Nature 256, 495 to 497, 1975 .

The specification of the products obtained is made using of the RIA test by measuring the binding capacity of a

1 25
JK antigen determined.

The hybrids that respond positively to the test are then isolated and cloned.

0 Large amounts of anti-K monoclonal antibody are produced by received intrapertional injection into BALB / c mice of cells secreting the anti-K monoclonal antibody.

The growth of the tumor, caused by the injected 3b cells, shows itself as the formation of ascitic fluid, which contains up to 20 mg / ml anti-K monoclonal antibodies. One works in an analogous way, but one uses

kn
for the immunization P- or P -antigenic determinants instead of the K-antigenic determinants, one obtains anti-

k η

P -monoclonal antibodies or anti-P -monoclonal antibodies.
5

Example 9

A solution of K-antigenic determinants, prepared according to the method of Example 3, (10 μg) in pH 7.2 PBS (20 μΐ) is for 5 min in an ice bath with a solution

ι 25
of Na I (1 mCi) in PBS (10 μΐ) and a solution (20 μΐ) which contains Chloramine-T in PBS in a concentration of 0.5 mg / ml.

Then 20 μΐ of a solution containing sodium metabisulfite in PBS at a concentration of 2 mg / ml, and 20 μΐ of a solution containing potassium iodide in PBS with a concentration of 20 mg / ml is added to the mixture. The reaction product is purified by HPLC to free J, and so J-radioiodinated K-

1 25
antigenic determinants (JK) obtained quantitatively.

According to an analogous process, radioiodinated P -

125 k k

own determinants (J-P) and radioiodinated K / P -anti-

gene mixtures ( ¹²⁵ JK / P ^k ) produced.

Example 10

Human serum samples from 335 hospitalized patients with non-neoplastic diseases are collected using the Anti-K antiserum obtained according to Example 6 using the reagents given below and analytical procedures checked.

Reagents

(1) Comparison buffer, which contains:
pH 7.2 phosphate buffer 70 mM,

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Sodium chloride 70 mM,
Sodium azide 31 mM and
Beef albumin 0.4% w / v.

(2) Anti-K antiserum, obtained according to Example 6, suitably diluted 1: 500 00 in:
pH 7.2 phosphate buffer 70 mM,
Sodium chloride 70 mM, j

EDTA disodium salt 33 mM, |

Sodium azide 31 mM and '<

normal goat serum 0.1% v / v. j

125 k '

(3) Radioiodinated label which is a J-K / P antigen j

Mixture, diluted in the buffer (1), contains so that a concentration of 1.5 ng / ml and a specific Activity of 60 mCi / mg are present.

(4) Second antiserum: antiserum precipitating goat immunoglobulins, which were bred in rabbits and appropriately diluted in the buffer (1).

(5) Negative control: normal human plasma.

(6) Positive control: normal human plasma to which one known amount of K / P antigen mixture was added.

Analytical procedure

"Zero" negati- positive sample ver comparison comparison equal comparison (hbuffer (1) 0.5 ml - -

negative comparison (5) - 0.5 ml

positive comparison (6) - - 0.5 ml

Sample - - - 0.5 ml

Anti-K antiserum (2) 0.2 ml 0.2 ml 0.2 ml 0.2 ml 35

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Mix, incubate, 5 h at room temperature.

Radioiodinated marker (3) 0.1 ml 0.1 ml 0 ,1 ml 0 ,1 ml 5 Incubate overnight at Room temperature Second antiserum (4) 0.2 ml 0.2 ml 0 , 2 ml 0 , 2 ml

Incubate for 1 h at room temperature, centrifuge for 30 min at 4000 rpm, decant and determine the radioactivity in the precipitate using a gamma counter.

Calculation: The PI-positive index is calculated by measuring the radioactivity of the samples or comparisons and relating them to the radioactivity of "zero" according to the following equations:

PI. (Sample) = 100 - ,, x 100

Activity of "zero

_,, ... Λ activity of comparison _inn

PI (comparison) = 100 - - r ^ -xr - »x 100

³ activity of "zero

A P.I. index value higher than 15 was considered indicative of the Presence of adenocarcinoma is considered, while a P.I. index value below 15 indicates the absence of one Adenocarcinoma was considered.

The above 335 samples were also tested in parallel using two commercially available kits for the detection of human CEA, namely the CEA-Roche test, and the Liso-Pha'se-CEA kit from Lepetit.

The results obtained are summarized in the following table .

Number of pathologies false positive results

the Roche Lepetit invention

Cases test according to test

30 cirrhosis of the liver 4 (13.3%) 3 (10%) 2 (6.6%) 17 inflammatory

Lung disease4 (23.5%) 4 (23.5%) 2 (11.7%) 18 obstructive

Bronchial pneumo-

pathia 3 (16.6%) 3 (16.6%) 1 (5.5%) 270 other non-

neoplastic

Diseases 4 (1.5%) 5 (1.8%) 1 (0.3%)

335 Total 15 (4.5%) 15 (4.5%) 6 (1.8%)

Example 11

Human serum samples from 84 patients who were diagnosed at. Suffering from adenocarcinoma, are analyzed using the reagents and analytical method as given in the example 0 10 are described, and by using the two commercially available kits mentioned in Example 10 in parallel were used.

The results are summarized in the following table.

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Number of pathologies false negative results

the Roche Lepetit invention

Cases test according to test

60 gastrointestinal adenocarcinomas 5 (8.3%) 7 (11.6%) 2 (3.3%) 24 pulmonary adenocarcus

name 2 (8.3%) 2 (8.3%) 0 (0%)

84 Total 7 (8.3%) 9 (10.7%) 2 (2.4%)

example

The same human plasma samples from Examples 10 and 11 are also checked by checking the reagents and alternative analytical methods listed below were used.

Reagents

(1) Anti-K antibodies obtained according to the method of which in Examples 5 and 6, and absorbed on polystyrene beads.

(2) Dilution buffer containing: pH 7.2 phosphate buffer 7 0 mM, Sodium chloride 70 mM, sodium azide 31 mM and Beef albumin 1% (W / V)

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

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

(5). Blpc ^ iiSiru ^ gsitrifct '^ !: 1M Sodium Carbonate.

(6) Negative control: normal human plasma.

(7) Positive control: normal human plasma to which a known amount of K / P antigen mixture has been added,

Analytical procedure

"Zero" negati- positive sample

ver comparison comparison

Dilution buffer (2) 0.2 ml 0.1 ml 0.1 ml 0.1 ml

negative comparison (6) - 0.1 ml

positive comparison (7) - - 0.1 ml

Sample - - - 0.1 ml

Anti-K Antibodies (1) 1 bead 1 bead 1 bead 1 bead

Incubate with shaking for 5 h at room temperature. Liquid phase is discarded.

Wash twice with 2 ml of saline solution.

Enzymatic conjugate

(3) 0.2ml 0.2ml 0.2ml 0.2ml

Incubate with shaking for 2 hours at room temperature. Discard the liquid phase.
Wash twice with 2 ml of saline solution.

30th Enzymatic substrate
(4) 1 h 1 ml 1 ml 1 ml 1 ml Incubate during (5) at 37 ⁰ C. t ■ 35 Blocking agents 1 ml 1 ml 1 ml 1 ml

Determined absorbance at 4 20 nm using a spectrophotometer.

calculation

The positive index P.I. is based on the optical Density (O.D.) of "zero" according to the equation

PD of the sample
^{p> 1} · "OD of" zero "

certainly. A positive index value above 3 is considered an indication of the presence of adenocarcinoma, whereas a positive index value below 3 is considered an indication of the absence of adenocarcinoma.

Results similar to those reported in Examples 10 and 11 were obtained.

Example 13

Tissue sections fixed with formalin and embedded in paraffin of human colon adenocarcinomas, human mammary adenocarcinomas and bone marrow from patients with breast 0 adenocarcinoma are analyzed using the following reagents and analytical procedures described.

Reagents

(1) Wash buffer, which contains:
pH 7.3 phosphate buffer 10 mM and

Sodium chloride 150 mM.

(2) Blocking buffer, which contains: pH 7.3 phosphate buffer 10 mM,

Sodium chloride 150 mM and

normal donkey serum 3% (V / V).

(3) Enzymatic conjugate which is anti-goat immunoglobulin antibody, bred in a donkey and labeled with ß-galactosidase (according to the procedure outlined in FEBS Letters 95, 311, 1978), and which is appropriately diluted in buffer (1).

(4) Enzymatic substrate, which contains:

pH 7.4 phosphate buffer 10 mM,

Sodium chloride 10 mM,

Magnesium chloride 1 mM,
5-bromo-4-chloroindolylgalactoside 1 mM, potassium ferric cyanide 6 mM and

Potassium ferricyanide 6 mM.

(5) Contrast dye, which contains: aluminum potassium sulfate and

Carminic acid.

(6) Anti-K antibodies raised in a goat and obtained by following the procedure of Example 7 and appropriately diluted in the buffer (1) above.

Analytical procedure

The sections were in xylene, in 100% to 50% ethyl alcohol series and then dewaxed in washing buffer (1). The reagent was added in such an amount that the entire section is covered.

Blocking buffer (2).

Incubate for 2 0 min at 37 ⁰ C in a humid chamber. Eliminate excess reagent by shaking.

0 addition of anti-K antibodies (6).

Incubate for 2 h at 37 ⁰ C in a humid chamber.

Addition of enzymatic conjugate (3).

Incubate for 30 min at 37 ⁰ C in a humid chamber. Wash the section with the buffer (1) for 5 min.

Add the enzymatic substrate (4).

Incubating for 3 0 min the excess reagent by washing at 37 ⁰ C. eliminating.

Adding the contrast dye (5).

After 1 min, the section was washed in running water and observed using a light microscope.

1.0

In an alternative procedure, the above analytical procedure was performed, with the incubations overnight were carried out at room temperature in a humid chamber, except for the incubation with the enzymatic substrate (4), which was carried out for 1 hour at room temperature in a humid chamber.

Always as an alternative to the above procedure, after the last wash with running water and before the test Using the microscope, first pass the sections through a wash buffer (1) containing 50% to 100% ethyl alcohol and xylene cleared and then on. Eukitt attached.

Microscopic analysis of the sections reveals an intense blue discoloration that is exclusive to the cytoplasmic and of the membrane of the tumor cells is localized without Ir-.any positive discoloration reaction in the cells of the neighboring one normal tissue such as granulocytes.

It has been found that these results are in direct contrast to those obtained by using the same preparations using the commercially available kits for the histochemical detection of the CEA complex via anti-CEA antibodies, namely the DAKO Pap Kit and the Immunolok Histoset Kit.

^f COPY

Indeed, the commercially available reagents show very poor specificity since they have been found to be discolour not only the tumor cells but also the normal tissue cells to varying degrees, whereby the Error is particularly high in the analysis of bone marrow tissues.

The same procedure as described above was used for the analysis of frozen tissue sections and were obtained analogous results.

Example 14

The doxorubicin was conjugated to the anti-K antibodies using a covalent linkage process.

A solution of doxorubicin (40 mg / ml) in pH 7.2 0.015M Phosphate buffered salt comes with a slightly small excess of 0.1M NaJO. mixed and for 1 h at room temperature incubated in the dark.

Towards the end of the incubation, 1M glycerol is added to a final concentration of 0.05M.

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

0 Towards the end of the incubation, NaBH. added up to the final concentration of 0.3 mg / ml and the reaction can proceed ^{at 37 ° C. for 2 h.}

Towards the end of the reaction, the mixture is passed on to a gel filtration column (15 χ 1.5 cm) of Biogel P 100, equilibrated in PBS, chromatographed.

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The fractions in the collected volume contain the Drugs bound to the antibody and not a non-conjugated drug.

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Claims (1)

KRAUS & WEISERT 3337/55 PATENT LAWYERS ' AND APPROVED REPRESENTATIVES BEFORE THE EUROPEAN PATENT OFFICE DR. WALTER KRAUS D IPLOMCH EM IKER. DR.-IN G. ANN EKÄTE WEIS ERT DIPL-ING. I = ACH DIRECTION CHEMICALS IRMGARDSTRASSE 15 · D-8OOO MUNICH 71 · TELEPHONE O89 / 7O7O77-7n7 () 78 · TELEX OS-912 U>«> kpril il TELEGRAM CRAUS PATENT 3986 AW / rm FARMITALIA CAREO ERBA S.p.A. Milan / Italy New antigenic determinants related to adenocarcinoma, antibodies specific for them, methods for their preparation and reagent systems containing them Claims 1. One suitable for the diagnosis of human adenocarcinomas Method by determining the antigenic material related to the adenocarcinoma in a sample, taken from the human patient, characterized in that "using determined by (a) anti-K antibodies or (b) anti-P antibodies material in the sample which [a. ') exclusively contains those antigenic determinants K which are specific to the adenocarcinomas, or (b ¹ ) such antigenic determinants P associated with the K determinants on adenocarcinomas determined. COPY 2. The method according to claim 1, characterized in that the removed from the human patient Sample is a sample from a biological fluid or from tissue extract or a section. 3. The method according to claim 1 or 2, characterized in that - indicates that the determination of K- or P- antigenic determinants are carried out qualitatively, quantitatively or semiquantitatively.
10 4. The method according to any one of the preceding claims, characterized in that monoclonal anti-K antibodies or anti-P monoclonal antibodies can be used. . 5. Anti-K antibodies responsible for the K antigenic determinants are specific. 6. A method for the production of anti-K antibodies according to claim 5, characterized in that the antibodies formed against a mixture of K and P antigenic determinants with P ⁿ - or P antigenic determinants are absorbed, so that the P -immunological binding sites on the antibodies become saturated. 25th 7. A method for the production of anti-K antibodies according to claim 5, characterized in that one η k 1) Animals with P or P antigenic determinants below η k Immunized formation of anti-P or anti-P antibodies, 2) a mixture of antigenic determinants K and P with- n k by means of anti-P or anti-P antibodies, which according to 1) were prepared, purifies so that K-antigenic determinants are obtained, and 3) animals, including mammals, with K-antigenic determinants, which have been obtained according to 2), immunized. k k 8. Anti-P antibodies responsible for P antigenic determinants are specific. 9. Process for the preparation of anti-P antibodies according to claim 8, characterized in that one Animals, including mammals, immunized with P antigenic determinants. 10. Anti-P ⁿ antibodies specific for P ⁿ antigenic determinants. 11. Process for the production of anti-P ^ antibodies according to Claim 10, characterized in that one Animals, including nanten immunized. Animals, including mammals, with P -antigens Dclormi-. Π 2J Antigenic determinants K specific for human adenocarcinomas. 13. Process for the production of K-antigenic determinants. ten according to claim 12, characterized., that a mixture of antigenic determinants K and I 'init- n k using anti-P or anti-P antibodies. 14. Antigenic determinants P, thereby g e k e η η - shows that the antigenic determinants P are those antigenic determinants with those of the K-antigenic determinants are associated with human adenocarcinomas. 15. A process for the preparation of P antigenic determinants according to claim 14, characterized in that a mixture of antigenic determinants K and P ^{v is} purified by means of anti-P antibodies. 'BAD ORIGINAL 16. ■. Antigens, - determinants P ⁿ , characterized in that the antigenic determinants P ^{n are} those antigenic determinants which are present in normal plasma or serum and which result in a cross-reaction with the P -antigenic determinants. 17. Process for the production of P -antigenic determinants according to claim 16, characterized in that normal plasma or serum is extracted and then purifies by immunoaffinity with specific antibodies. - ' 18. Monoclonal anti-K antibodies against the singular K-antigenic determinants are specific. -. 19. Anti-P monoclonal antibodies opposite to the singular P antigenic determinants are specific. 20. Anti-P monoclonal antibodies ^{specific to the singular P n} antigenic determinants. 21. Process for the production of monoclonal antibodies • according to claim 18, 19 or 20, characterized ', that the monoclonal antibodies are obtained according to the hybridoma technique. 22. Reagent system for use in a method for diagnosing human adenocarcinomas according to any one of the claims 1 to 4, characterized in that it contains anti-K antibodies as an essential component, which are specific to the antigenic determinants K. 23. Reagent system according to claim 22, characterized in that g e k e η η that it contains anti-K monoclonal antibodies. 24. Reagent system for use in a procedure copy - ~ , Dalj enth.'ilt as an essential component anti-P antibody for the diagnosis of human adenocarcinomas according to any one of claims 1 to 4, characterized -ge ^v ke η η ζ eichhet it that. are specific to the antigenic determinants p '. 25. reagent system according to claim 24, dadurcli g e k e η η - ζ e i holds. Xr records that there are anti-P monoclonal antibodies ent 26. Reagent system for use in a radioimmunoassay procedure for the diagnosis of human adenocarcinomas according to one of claims 1 to 4, characterized in that it essentially k
1) anti-K or anti-P antibodies, 2) ' radiolabeled K or P antigenic determinants or a
mix, k or alternatively a radiolabeled K / P antigenic Ge 20th 3) Compare and 4) buffer
contains. 27. Reagent system according to claim 26, characterized in that there is a secondary component as a further component Antibodies against immunoglobulins of the species Ir zie in which the anti-K or anti-P antibody of component 1) was grown, specifically contains. 28. A reagent or 27, characterized in any one of claims 26, that the radiolabelled component 2) JK ¹²⁵ or ¹²⁵ or ¹²⁵ jp ^k JK / P ^k. 29.Reagent system for use in an enzyme immuno- COPY] assay method for the diagnosis of human adenocarcinomas according to one of claims 1 to -4, characterized in that it is essentially 1) Anti-K or anti-P antibodies that attach to a solid Phase are bound, 2) enzyme-conjugated anti-K or anti-P antibodies, 10'3) an enzymatic substrate, 4) Compare and 5) buffer ■ 15 '■■ ·';'· ^: ' - contains. 30. Reagent system according to claim 29, characterized in that g e k e η η zei chnet that the enzyme of component 2) ß-galactosidase and that the enzymatic substrate of component 3) contains o-nitrophenylgalactopyranoside. 31. Reagent system for use in an immunohistochemical Method for the diagnosis of human adenocarcinomas. according to one of claims 1 to 4, characterized in that g e k e η η that it is essentially j,
1) Anti-K or anti-P antibodies, 2) enzyme-labeled antibodies which are specific to immunoglobulins of the species in which the anti-K or anti-P
have been tef, 3) an enzymatic substrate and
4) buffer COPY or or anti-P antibodies of component 1) bred contains. ^ ₁ . · 32. Reagent system according to claim 31, characterized in that the enzyme of component 2) ß-GaIactosidase and that the enzymatic substrate of component 3) contains 5-bromo-4-chloroindolylgalactoside. 33. Labeled K, P and P ⁿ antigenic determinants or k η labeled anti-K, anti-P and anti-P polyclonal or monoclonal Antibody. 34. Labeled antigenic determinants or antibodies according to claim 33, characterized in that the antigenic determinants and antibodies are radioactively labeled. 35. Labeled antigenic determinants or antibodies according to claim 33, characterized in that the antigenic determinants and antibodies are erizyme-labeled. . 36. Anti-K or anti-P polyclonal or monoclonal antibodies for use as carriers of anti-tumor drugs or cytotoxic agents in the therapy of human Adenocarcinoma. - 37. Conjugates between a polyclonal or monoclonal anti-K or anti-P antibody and an anti-tumor drug or a cytotoxic agent. 38. · Conjugates according to claim 37, characterized in that the antitumor drug is selected is taking daunorubicin, doxorubicin, epirubicin, and methotrexate. 39. Conjugates according to claim 37, characterized in that the toxic agent is ricin. COPY,