EP0197974A1

EP0197974A1 - Acino-foetal differentiation proteins associated to the pancreas cancer, antiserum and monoclonal antibodies to said proteins, preparation methods

Info

Publication number: EP0197974A1
Application number: EP85904826A
Authority: EP
Inventors: Maria Juana Escribano-Crespo; Pierre Burtin
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: ETABLISSEMENT PUBLIC DIT: CENTRE NATIONAL DE LA R
Priority date: 1984-10-01
Filing date: 1985-09-30
Publication date: 1986-10-22
Also published as: OA08334A; JPS62500304A; WO1986002081A1; FR2571146A1; EP0180496A1; FR2571146B1; DE3570677D1; ATE43610T1; US4843019A; EP0180496B1

Abstract

PCT No. PCT/FR85/00268 Sec. 371 Date May 29, 1986 Sec. 102(e) Date May 29, 1986 PCT Filed Sep. 30, 1985 PCT Pub. No. WO86/02081 PCT Pub. Date Apr. 10, 1986.The invention relates to acino-fetal differentiation proteins associated with cancer of the pancreas, these proteins being mannoside-containing glycoproteins of average apparent molecular mass chosen from: 120 KD, 94 KD and 58 KD. The invention also relates to a method for preparing these proteins, an antiserum against these proteins and the method for preparation thereof, monoclonal antibodies against these proteins and the method for preparation thereof, and compositions for diagnostic or therapeutic use containing these proteins or antibodies.

Description

ACINOFOETAL DIFFERENTIATION PROTEINS ASSOCIATED WITH PANCREAS CANCER, ANTISERUM AND ONOCLONAL ANTIBODIES AGAINST THESE PROTEINS, METHODS OF PREPARATION.

The present invention relates to acino-fetal differentiation proteins associated with pancreatic cancer, their purification, a specific antiserum and its preparation, monoclonal antibodies specific for these proteins and their preparation process as well as the compositions for diagnostic use and for therapeutic use containing them.

The demonstration of the presence, in extracts of fetal pancreas, of antigens analogous to those of the pancreatic tumor cells is known.

Human oncofetal pancreatic antigens have been identified, having a molecular weight of 40 KD (P0A-1) (banwo O., Versey J. and Hobbs JR, New ncofetal antigen for human pancreas. Lancet, 1: 643-645, 1974; Hobbs JR, Knapp, ML and Branfoot AG Panereatic oncofetal antigen (POA): its frequency and localization in humans. Oncod. Biol. And Medicine, 1: 37-48, 1980), 800 and 900 KD (POA-2) (Gelder FB , Resse CJ, Moossa AR, Hall T. and Hunter R. Purification partial characterization and clinical evaluation of a pancreatic oncofétal antigen. Cancer Res. 38: 313-324, 1978) and 1000 KD (ABCP) (Shimano T. , Loor RM Papsidero LD et al. Isolation, characterization and clinical evaluation of a pancreas cancer assoclated antigen. Cancer., Supplement, 47: 1602-1620, 1981). However, none of these antigens is specific for pancreatic cancer because they are found in tissues different from pancreatic cancer tumors.

The present invention relates to proteins of differention-fetus acion associated with pancreatic cancer, characterized in that they are manosid glycoproteins, of apparent molecular πpyenne mass chosen from: 120 KD, 94 KD,

75 KD and 58 KD.

The present invention also relates to a method

10 for the preparation of these proteins, characterized in that a homogenate of soluble human fetal pancreas is prepared and that a fraction consisting of manoside glyco¬ proteins is isolated.

The process for preparing these antigens includes

The following stages: a) a homogenate of soluble human fetal pancreas is prepared, b) the homogenate is treated by chromatography on an affinity column on concanavalin A coupled to a gel

In order to fix said proteins, c) the proteins in question are eluted, and d) optionally, the proteins obtained are purified. The preparation of the homogenate is described in the example

1.

25 Young fetuses less than 6 months pregnant, preferably less than 5 months, are used because the proteins according to the invention do not appear in the adult pancreas and only rabbits injected with the pancreas of the fetus older than 6 months did not produce anti

30 detectable bodies.

In a particular embodiment, Sepharose gel 4B was used coupled with concanavalin A, marketed by Pharmacia. The unbound fraction is eliminated, the fixed fraction is eluted, using a specific ligand, ar

35 ex-, α-methylmanoside 0.2 M. We then align the eluted fixed fraction by a conventional technique and the proteins are separated, for example by preparative electrophoresis. The peaks corresponding to the proteins according to the invention have previously been identified by qualitative electrophoresis. The acrylamide gel fractions loaded with proteins according to the invention are then removed, and the proteins are extracted therefrom by electroelution in a dialysis hose against a PBS phosphate buffer (0.15 M saline solution buffered NaCl at pH 7 with final 0.01 M potassium phosphate buffer). It is dialyzed against distilled water and lyophilized. A characterization of the proteins according to the invention is described in Example 6.

To separate the differentiation proteins, it is also possible to use the techniques of fixation on the monoclonal antibodies. The present invention also relates to an antiserum against the antigens according to the invention.

By the "blotting" technique (after acrylamide gel electrophoresis - PAGE -, the proteins which have migrated in the gel are electrophoretically transferred to a nitrocellulose sheet; this sheet is then treated as in the NIF technique.) , this antiserum recognizes, in fetal pancreas extracts, 4 constituents of average apparent molecular mass 120, 94, 75 and 58 KD (to within 10%). The present invention relates to spreading a process for the preparation of this antiserum comprising the following steps: a) an animal is injected with an extract of human fetal pancreas less than 6 months old, b) the animal is bled and an antiserum is recovered , c) this antiserum is decomplemented, d) the decomplementary antiserum is absorbed, e) the absorbed decomplementary antiserum is centrifuged, and f) the specific antiserum is recovered. Preferably, the animal is injected with an extract of the fetal pancreas less than 5 months old.

This antiserum is prepared according to the protocol described in Example 2 against crude pancreatic extracts prepared as described in Example 1. In the particular embodiment described in Example 1, only the grooves No. 4 and 5 are positive in an antibody test. In one embodiment of the invention, the extract was injected into a rabbit. Mice and hens, for example, can also produce antiserum against these extracts.

To decomplement the antiserum, it is heated in a water bath at about 56 ° C for 1/2 hour, according to a conventional technique. The antiserum is then made specific to the fetal pancreas by absorption of various extracts, for example extracts from normal adult pancreas, normal serum, red blood cells A, O, B, and possibly other organs.

For this, 2 ml of normal serum is added per ml of antiserum and 250 mg of normal adult freeze-dried pancreas extracts per ml of antiserum.

To obtain the normal adult pancreas extract, the procedure is as in Example 1, the pancreas comes from donor young people who died accidentally.

The decomplemented antiserum absorbed is purified by a conventional centrifugation technique.

The purified absorbed antiserum was tested for its ability to detect antigens by two techniques:

A) Immunofixation on nitrocellulose membranes

(NIF technique) This technique is described in 1 * example 3. It makes it possible to detect very small quantities of one or more antigens in complex mixtures, for example extracts gross organs, pathological seru s. This technique can detect approximately up to 0.1 μg of differentiation proteins per ml in crude organ extracts or body fluids.

The sensitivity varies with the antigen-antibody system and can go up to 0.01 μg of antigens per ml, or 0.0005% when, as for the tests collected in table 1, the extracts are analyzed from 20 mg / ml.

By this technique, the antiserum was positive for fetal pancreatic extracts, pancreatic tumors and liver metastases from primary pancreatic cancer. However, other extracts from adult organs were negative. or fetuses detailed in Table I.

TABLE I

EXTRACTS FROM NORMAL ORGANS AND NORMAL SERUM

Pancreas + Adult Fetal Organs

Liver Stomach Lung

Intestine - -

Kidney Rate

Normal serum - -

. The extracts are "pools" of organs used in decreasing concentration from 20 mg / ml.

. N .T. = Not Tested

By this same technique, 100% of the sera of patients with pancreatic cancers were positive (N = 17) while the serum of patients with other tumors was negative (see Table II).

TABLE II

PATHOLOGICAL SERUMS

Serun Pathological Noβbre tested Positive number8

Pancreatic cancer 17 17

"stomach 2 0

"colon 2 0

"lung 2 0

"kidney 2 0

. The sera are used at lg2 dilutions from the normal concentration.

B) Immunohistology on sections fixed with ethanol or Bouin's liquid The method used is described in Example 4. Using either the immunofluorescence technique or indirect immunoperoxidase, the antiserum marks the fetal acinar cells up to at around the 6th month of gestation and is negative for other fetal or adult organs. It has marked all pancreatic cancers analyzed and has been negative for a wide variety of other types of cancer. The results of the immunohistology are detailed in Table III.

TABLE III

Reaction of the fetal anti-pancreatic antiserum in NIF and immunohistology.

Cancer Number tested Positive Negative

Pancreas 18 18 0

Liver 11 0 11

Stomach 8 0 8

Colon 10 0 10

Salivary glands 4 0 4

Lung 4 0 4

Bladder 6 0 6

Gallbladder 6 0 6

Breast 3 0 3

Prostate 7 0 7

Normal fabrics

Fetal pancreas 20 18 2 (±) 6 months and under

Fetal pancreas 16 8 8 over 6 months

Adult pancreas

Duodenum

Stomach

Gallbladder (canals)

Salivary glands

Chronic panereatitis 11 Chronic pancreatitis is a special case because they are generally negative, but in some cases we observe a marking similar to that found in the p ritumoral areas. The antiserum according to the invention therefore makes it possible to detect serum and tissue levels which are too low to be assayed by precipitation, the sensitivity threshold of which is around 10 μg / ml.

The Applicant has demonstrated that the proteins according to the invention appear not only in pancreatic tumors, but also in pretumoral lesions. These differentiation proteins therefore also have the value of pretumoral markers.

Indeed, during the experimental chemical carcinogenesis in the hamster, they are expressed around the second month whereas the first tumors are not detected in histology until around the 8th month.

In humans, these antigens express it intensely not only in cancers but also in pretumoral markers.

The following table presents other analysis results showing the specificity of these differentiation proteins for other normal fetal and adult tissues, by NIF and immunohistology techniques.

10

TABLE IV

Adult Fetal Tissue NIF Hist NIF Hist

Pancreas +

Liver -

Lung -

Stomach -

Colon -

Small intestine -

Salivary glands NT NT NT

Bladder II

Gallbladder II

Muscular -

Skin - NT NT

Brain -

Missed -

Kidney -

Serum -

+ positive reaction; - negative reaction; NT not tested These differentiation proteins according to the invention can also be applied to the development of specific antibodies.

Thus, the Applicant has carried out the preparation of monoclonal antibodies specific for the differentiation proteins according to the invention.

This is why the present invention relates to monoclonal antibodies specific for proteins of apparent molecular mass differentiation 120 KD, 94 KD, 75 KD and 58 KD.

The present invention also relates to a process for the preparation of these monoclonal antibodies, characterized in that it comprises the following steps: a) a mouse is immunized with at least one of the differentiation proteins, b) a cell fusion of cells is carried out spleen of these mice, with an SP ~ myeloma, c) the hybridomas obtained are cultured in an appropriate medium, d) the cultures are screened, e) the hybridomas positive for screening are cloned and cloned specifically with the fetal pancreas, and, f) isolating and optionally purifying the monoclonal antibody. To prepare the antibodies, the conventional hybridization technique is therefore used, as developed by Kohler and Milstein in 1975, by immunizing mice, for example BALB / C mice with the fraction of a homogenate of fetal pancreas. under 6 months purified on Concanavalin A previously prepared. In a particular embodiment of the invention, differentiation proteins prepared according to the invention were used to immunize the mice from fetal pancreas 3 to 4 months old. For the screening, that is to say the selection of producer hybridomas, the NIF technique, already described, is used for example. The cultures are tested with various extracts, for example extracts from normal adult pancreas, normal adult and fetal serum, and an extract from fetal pancreas, in order to keep only the cultures which react specifically with the fetal pancreas. .

The monoclonal antibodies are then selected by conventional techniques. It is thus possible to use the "Immuno Blot" technique using SDS-PAGE (example 5), a technique also known by the name of "Western Blot" and described by Towbin et al. (Electrophoretic Transfer of proteins from polyacrylamides gels to nitrocellular sheets procedure and some application, Prot. Nat. Acad. Sci. USA 7_, 1979 page 350-354).

The monoclonal antibodies are then isolated in the supernatant of the cultures of selected hybridomas, by a usual technique.

Monoclonal antibodies can also be obtained from ascites by injecting the producing cell, monoclonal antibody, into mice.

The Applicant has thus isolated two monoclonal antibodies specific for the differentiation protein according to the invention with an average molecular mass of 120 KD.

By typing, it is demonstrated that these are Ig G type antibodies.

The antibodies can then be isolated by affinity chromatography on protein A fixed on sepharose, for example, eluting at a pH between 3 and 6. The present invention therefore also relates to compositions for diagnostic use containing, as active element, at least one differentiation protein or a monoclonal antibody according to the invention and an acceptable substrate, for example in the form of immunodiagnostic kits. according to a mechanism based on antibody-antigen reactions.

The differentiation proteins and monoclonal antibodies according to the invention are also useful as specific tumor and pretumoral markers which can be used for imaging.

This is why the present invention relates to compositions for diagnostic use comprising the differentiation proteins or the monoclonal antibodies according to the labeled invention.

For this use, the labeling can be carried out either by any marker, in particular fluorescent, radioactive or paramagnetic markers.

Finally, the present invention also relates to the compositions for therapeutic use comprising at least one differentiation protein or a monoclonal antibody according to the invention conjugated to an active principle.

EXAMPLE 1

Preparation of fetal pancreatic extracts From fetuses in good condition, after therapeutic abortion, at 3 to 4 months of gestation, the pancreas which is rapidly immersed in an antiprotease solution containing aminocaproic acid (0.4 %) and aprotinin (40 units of Kallikreine / ml), for example aprotinin Trasylol ^ sold by Sigma. The pancreas, at a rate of 100 mg / ml of antiprotease solution, is ground as soon as possible after removal with a Ultraturax mill for a few minutes in the cold.

The extract is centrifuged at 20,000 rpm for 1/2 hour, the supernatant is aliquoted and stored at -80 ° C.

These extracts contain between 5 and 8 mg of protein per ml. EXAMPLE 2

Antiserum preparation The above-described pancreas extract is injected into a rabbit according to the following protocol:. Day 1: 1 ml of extract + 1 ml of complete Freund's adjuvant in intradermal injections on several points on the back. . Day 24: Same operation as Day 1.. Day 44: Test bleed n ° 1.. Day 54: Re-immunization as Days 1 and 24.. Day 78; Test bleed # 2.

. Day 98: New injection as before. . Day 110: Test bleed n ° 3.

. Day 150: Intravenous injection without adjuvant (clear centrifuged solution). . Day 156: Test bleed n ° 4.

. Day 186: New intravenous injection.

. Day 192: Tapping n ° 5.

The serum is then decomplemented, by bringing it to a water bath at 56 ^β C for approximately 30 minutes, then absorbed by extracts of normal adult pancreas, normal serum and red blood cells A, 0, B.

EXAMPLE 3

NIF technique. Cut a piece of nitrocellulose of appropriate dimensions (nitrocellulose membranes BA85,

Schleicher and Schiill - Dassel, R.D.A.).

. Deposit the solution in which the antigen is sought in microdrops of 1 to 5 μl. The concentration is chosen according to the desired sensitivity. The dilutions of the antisera are made in a solution

2% egg white ovalbumin, in distilled water, sold by Sigma.

. Let dry. Rinse with P.B.S. . Incubate for 1 hour at 4 ° C in a saturated protein solution (2% egg white ovalbumin solution, in distilled water, marketed by Sigma) to block the binding sites of the membrane not occupied by l 'antigen. . Rinse with P.B.S. Incubate with antiserum.

In this case, for absorbed fetal pancreatic serum, it is used diluted 1/1000 overnight at 4 ° C.

. Wash the filter thoroughly to completely remove the antiserum. . Incubate with anti-rabbit IgG antibody conjugated to peroxidase (Institut Pasteur Production), diluted 1/2000 for 2 hours at 4 ° C. . Wash again thoroughly with PBS

. Reveal the enzymatic reaction by incubation in the peroxidase substrate: H.O. 0.01% and the chromogen .; This can be either chloronaphthol. (Sigma): blue coloration, i.e. 3.3 '-diaminobenzidine (: D.A.B., Merck): brown coloration. The first is dissolved at 3 mg / ml in methanol and then diluted 5 times in P.B.S. The second (D.A: B.). Es. I directly in the P.B.S. (0.5 mg / ml). In both cases, add hydrogen peroxide with a final concentration of 0.01% as indicated above.

. When the reaction has developed (color stabilization), wash with distilled water and for

_2 D D..AA..BB .. ,, fix the color - by washing with HC1 10 M. EXAMPLE 4

Immunohistology

Organ sections, fixed either with ethanol, formaldehyde or Bouin's liquid, are included in the paraffin. Deparaffinize, hydrate and inhibit endogenous peroxidase.

1) Incubate with 2% bovine serum albumin (B.S.A.) in distilled water, 1 hour at 4 ° C.

2) Incubate with anti-fetal pancreatic antiserum diluted 1/25 in B.S.A. 1%, 4 hours, at 4 ° C. 3) Wash 3 times 10 minutes in P.B.S.

4) Incubate with anti-rabbit IgG antibodies conjugated to peroxidase (Institut Pasteur Production) diluted 1/100 in P.B.S.

5) Wash 3 times 10 minutes in PBS 6) Reveal the enzymatic reaction in a bath of 0.01% H ₂ O and aminoethylcarbazole. EXAMPLE 5 Immunoanalysis on nitrocellulose stains after SDS-electrophoresis on acrylamide gel.

The SDS-PAGE technique is used in 10% acrylamide gels in a Tris-glycine pH 8.3 buffer containing 0.1% SDS (Laenmli UK cleavage and structural proteins during assembly of head of the bacteriophage Nature (Lond. 227: 680-5 (1970)).

The nitrocellulose membranes are transferred to a Biorad transfer cell at 4 ° C. at 100 mA at constant current for 16 h at pH 8.3 (Towbin). The tasks are revealed as in the NIF technique.

EXAMPLE 6

Characterization of the differentiation proteins specific to the pancreas by the immunoblot technique after electrophoresis on acrylamide gel.

Homogenates of adult tumor and fetal pancreas (17, 20, 25 and 32 weeks) are analyzed by SDS-PAGE and are stained with Coomassie blue or transferred to nitrocellulose membranes. Later, the fetal anti-pancreas serum revealed several bands corresponding to the different differentiation proteins.

Based on the intensity of staining, most protein is almost absent in the 32-week pancreas.

The adult pancreas were not colored.

In a tumor, two proteins are present, while in another one is expressed mainly the protein of molecular mass 120 K. The evaluation of the intensity of the staining compared to the reference indicates that in all cases, the proteins - of differentiation. are present in small quantities compared to the total amount of protein in the crude homogenates.

Claims

1) Proteins of acdno-fetal differentiation associated with pancreatic cancer, characterized in that they are manoside glycoproteins, of average apparent molecular mass chosen from: 120 KD, 94 KD, 75 KD and 58 KD.

2) Process for the preparation of proteins according to claim 1, characterized in that a homogenate of soluble human fetal pancreas is prepared and that a fraction consisting of manosided glycoproteins is isolated.

3) Process for the preparation of acino-fetal differentiation proteins associated with .. pancreatic cancer from human fetal pancreatic extracts according to claim 2, comprising the following steps: a) a homogenate of fetal pancreas is prepared b) the homogenate is treated by chromatography on an affinity column on concanavalin A coupled to a gel in order to fix said proteins, c) the proteins in question are eluted, and d) optionally, the proteins obtained are purified.

4) Method according to claim 2 or 3, charac¬ terized in that the human fetal pancreas is less than 6 months old, preferably less than 5 months old.

5) Polyclonal antiserum against proteins according to claim 1, characterized in that it is specific for pancreatic cancer.

6) Process for the preparation of an antiserum according to claim 5, comprising the following steps: a) an animal is injected with an extract of the human fetal pancreas less than 6 months old, b) the animal is bled and an antiserum is recovered, - - c) this antiserum is decomplemented, d) the antiserum is absorbed decomplemented, e) the absorbed decomplemented antiserum is centrifuged, and f) the specific antiserum is recovered.

7. A method of preparing an antiserum according to claim 5, characterized in that an extract of human fetal pancreas of the fetus less than 5 months old is injected.

8. A method of preparing an antiserum according to claim 6 or 7, characterized in that it absorbs

1 * antiserum, in particular on red blood cells A, O, B, normal serum and / or normal pancreas.

9. A monoclonal antibody specific for a protein according to claim 1.

10. Method for preparing a monoclonal antibody specific for a protein according to claim 1, characterized in that it comprises the following steps: a) a mouse is immunized with at least one of the differentiation proteins, b) a cell fusion of spleen cells from these mice is carried out, with an SP- myeloma, c) the hybridomas obtained are cultured in an appropriate medium, d) the cultures are screened, e) the hybridomas posir are preserved and cloned tive in screening specifically with fetal pancreas, and f) _r isolated and possibly purified monoclonal antibody. 11) Method according to claim 10, charac¬ terized in that the differentiation proteins come from a fetus aged 3 to 4 months. 2) Composition for diagnostic use containing, as active element, at least one differentiation protein or a monoclonal antibody according to one of claims 1 or 9 and an acceptable substrate.

13) Composition according to claim 12, charac¬ terized in that the differentiation protein or the anti¬ body is labeled.

14) Composition according to claim 12 or 13, characterized in that it is a radioactive, paramagnetic or fluorescent labeling.

15) Composition for therapeutic use comprising at least one differentiation protein according to claim 1 or a monoclonal antibody according to claim 9 conjugated to an active principle.