WO1991001378A1

WO1991001378A1 - Monoclonal antibodies directed against the proenzymes of b-like cathepsine and their biological applications

Info

Publication number: WO1991001378A1
Application number: PCT/FR1990/000537
Authority: WO
Inventors: Daniel Keppler; Marie-Claude Fondaneche; Maurice Pagano; Pierre Burtin
Original assignee: Centre National De La Recherche Scientifique
Priority date: 1989-07-18
Filing date: 1990-07-17
Publication date: 1991-02-07
Also published as: FR2649891A1; FR2649891B1

Abstract

The monoclonal antibodies of the invention are directed specifically against the epitopes of PCBLs.

Description

MONOCLONAL ANTIBODIES DIRECTED AGAINST B-LIKE CATHEPSIN PROENZYMES AND THEIR BIOLOGICAL APPLICATIONS

The invention relates to monoclonal antibodies which recognize cathepsin B-like proenzymes, abbreviated as (PCBL).

Cathepsin B, abbreviated as (CB), is a lysosomal cysteine proteinase found in mammalian tissues. In the case of malignant tumors, the secretion of a proteinase of this type, called cathepsin B-like (CBL), is observed. But it is mainly the cathepsin B-like proenzymes (PCBL) that are secreted by cancer cells. PCBLs should also be studied in the case of immune deficiencies and in certain pathologies, notably inflammatory ones. PCBL maturation goes through several stages corresponding to different molecular weight forms, one from 45 to 47 kDa, the other from 36 kDa, CBL having a molecular weight of 33 kDa. We measure the interest of highlighting in these pathologies the presence of PCBL, in their different forms, before maturation of the CBL enzyme, in order to establish a diagnosis. The specificity of the reagents available to date for detecting and quantifying PCBLs in biological fluids and tissues is however not sufficient to establish high reliability diagnoses. Research by the inventors of more satisfactory reagents has led them, using PCBLs having a high degree of purity, to develop antibodies which give rise to immunological reactions only with PCBLs. The object of the invention is therefore to provide, as new products, monoclonal antibodies directed against PCBLs.

According to another aspect, the object of the invention is to provide the strains of hybridomas producing these antibodies.

According to yet another aspect, the invention aims to take advantage of the high specificity of these monoclonal antibodies vis-à-vis PCBLs to detect the possible presence of the latter in a biological sample, thus making it possible to diagnose the development of 'a pathology.

The monoclonal antibodies of the invention are characterized in that they have an elective specificity for PCBLs and that they recognize on these

PCBL epitopes which differ from antibody to antibody.

It follows from this elective specificity of the monoclonal antibodies of the invention that they neither react nor with mature enzymes; cathepsin B-like, cathepsins B, H and L, and papain, but selectively recognize one and / or the other form of PCBL.

Thus, the monoclonal antibodies of the invention are selectively directed against the PCBLs corresponding to the molecular weight forms of the order of 45-47 kDa and / or 36 kDa.

Such forms are described by Keppler et al in Biol. Chem. Hoppe-Seyler, vol 369, suppl. p.185-190, May 1988.

Thanks to their specific recognition properties for PCBLs, monoclonal antibodies can be used to quantify the different forms of these PCBLs. The expression "elective specificity" for

PCBL means that the monoclonal antibodies specifically bind to PCBL under the estern-blot (or dot-blot) and ELISA conditions described in Examples 1 and 2 below.

On the other hand, under these conditions, no reaction of the monoclonal antibodies of the invention is observed with the mature enzymes.

The monoclonal antibodies of the invention are further characterized in that:

- they belong to the class of IgGi-k,

- they have a molecular weight of approximately 150 kDa, and

- they have a high affinity for PCBLs. According to another aspect, the anti-PCBL monoclonal antibodies of the invention are characterized in that they are capable of being obtained by a process comprising the steps:

- immunization of an animal, according to conventional techniques, with purified PCBLs, fusion of animal cells producing antibodies directed against PCBLs with mouse yeloma cells,

- selection of hybridomas capable of producing specific antibodies against PCBLs,

- cloning of these hybridomas, and

- purification of monoclonal antibodies produced from ascites.

The immunization is advantageously carried out according to the technique described by Ternynck and Avrameas in Immunol. Rev. , 1986, vol. 94, p. 99-112. It is particularly interesting to use

Biozzi * which are good producers of antibodies.

For the fusion step, use is made on the one hand of cells from popliteal ganglia, on the other hand to myeloma cells SP2 / 0 of Balb / c mice.

Ascites are produced by injection of the clones into previously irradiated mice, for example Balb / c mice. The monoclonal antibodies recovered from ascites are purified by affinity chromatography. Satisfactory results are obtained using a Protein A-Sepharose ^R column.

The monoclonal antibodies according to the invention of great interest as diagnostic reagents are murine monoclonal antibodies produced by hybridomas resulting from the fusion of mouse SP2 / 0 myeloma cells with BI0ZZI ^R mouse popliteal lymph node cells, previously immunized with purified PCBL preparations.

Such antibodies include those produced by clones No. 1-890, 1-891 and 1-892 deposited on July 17, 1989 in the National Collection of Culture of Microorganisms (CNCM).

The invention also relates to the hybridoma strains producing the monoclonal antibodies defined above.

These hybridomas are characterized in that they are formed from hybrid cells resulting from the fusion of mouse myeloma cells with a mouse cell producing antibodies, after immunization of the mouse with purified PCBLs in form 36 and / or 45- 47 kDa, said hybrid cells being capable of producing antibodies specific for PCBLs.

The clones of antibody-producing hybridomas have the advantage of being homogeneous on the cellular level and of great stability.

More specifically, myeloma cells SP2 / 0 are used for the fusion and, as antibody-producing cells, lymph node cells. BIOZZI * mouse poplites, good antibody producers.

The preparation of these hybridomas also falls within the scope of the invention and comprises the steps of fusion and selection, defined above in relation to the hybridomas.

The specific properties of the monoclonal antibodies of the invention are used for the in vitro assay of PCBL in serum and other biological fluids, by various methods used in immunology, such as the RIA, IRMA or ELISA methods, in particular to detect cancers, inflammatory pathologies, immune deficiencies, or even other pathologies. To carry out a sandwich type immunoassay, two monoclonal antibodies reacting with two different epitopes of the same form of PCBL are used.

The second monoclonal antibody is coupled to a marker allowing the detection of the immunological reaction. It may be a chemical marker such as biotin capable of binding to a developer such as avidin peroxidase, or a radiolabel, such as radioactive iodine, or alternatively an enzymatic marker, such as than peroxidase, for example.

The method of in vitro detection of PCBL according to the invention also comprises: bringing a biological sample from a patient likely to be affected by a disease accompanied by the secretion of PCBL into contact with a monoclonal antibody as defined above, under conditions suitable for the production of an antigen-antibody complex, and - detection of the possible immunological binding of the monoclonal antibody to the PCBLs of the cells of the biological sample. The biological sample is a tissue preparation such as a cryostat cut for example. In general, the monoclonal antibody carries a marker as defined above. This diagnostic method is particularly useful in immunohistochemistry.

The method of in vivo detection by immunolocation of PCBL according to the invention comprises:

- an intravenous injection of a monoclonal antibody, as defined above carrying a radioactive marker making it possible to locate, by external scintigraphy or any other method, a malignant tumor or group of cells secreting PCBL, after immunological reaction between the labeled antibody and PCBLs.

In addition to their application as in vitro and in vivo diagnostic reagents, the monoclonal antibodies of the invention can be used as laboratory reagents for research purposes.

The invention also relates to a kit for detecting in vitro the presence of PCBL of 45-47 kDa in a biological sample according to the ELISA method. This kit is characterized in that it includes:

- an appropriate solid phase serving as a support for the assay, such as a micro-titration plate,

a preparation of monoclonal antibody specific for PCBL of 45-47 kDa according to the invention serving as first layer, a preparation of another monoclonal antibody reacting with another epitope of the form 45-47 kDa according to the invention serving second layer, this monoclonal antibody comprising a marker, - a specific detection system for the labeled monoclonal antibody;

- appropriate buffer solutions for immunological reactions and for detection reactions.

The properties of the monoclonal antibodies of the invention are also used in therapy, for the development of immunotoxins.

Immunotoxins are characterized in that they consist of monoclonal antibodies

- of the invention in association with a toxin.

In general, the invention relates to anti-PCBL therapeutic agents, produced from a monoclonal antibody as defined above, in association with a vehicle acharically

-5 acceptable.

Other characteristics and advantages of the invention will appear in the examples which follow. EXAMPLE 1: OBTAINING STRAINS OF HYBRIDOMAS PRODUCING ANTI-PCBL MONOCLONAL ANTIBODIES

-0 Biozzi mice (good antibody producers) are immunized by injection, into the plantar pad of the hind legs, of purified PCBLs according to the method described by Keppler et al (see reference above).

- Purified PCBL preparations include the 45-47 kDa and 36 kDa forms.

Two injections are made 20 days apart, with a preparation of PCBL containing complete Freund's adjuvant.

Five days after the last injection, the cells of the popliteal glands of these mice are fused with myeloma cells SP2 / 0 from Balb / c mice.

Three hybridomas 3-A, 3-B and

35 3-C which are cloned. Ascites are produced in Balb / c mice previously irradiated with a dose of 400 rad X-rays, by injecting the clone cells, then the βonocloπaux antibodies are purified on a column of Protein A-Sepharose.

In the table below, the results obtained with clones 3A2, 3B1 and 3C2 are reported, relating to the immunological reactions with the purified PCBLs.

SPECIFICITY CLASS EPITOPES

By the Western-blot technique after electrophoresis in polyacrylaid + SDS gel, it is found that clones 3A2, 3B1 and 3C2 react specifically with PCBLs: clone 3A2 reacts with the 36kDa form, and clones 3B1 and 3C2 with the form 45kDa.

Using the ELISA method and by dotblot, it was observed that the binding of one of these labeled monoclonal antibodies, for example by biotin, was only inhibited by the same unlabeled antibody, this for the antibodies monoclonals 3A2, 3B1, and 3C2. The monoclonal antibodies, 3A2, 3B1, and 3C2 are therefore directed against different epitopes.

By operating according to this example, it is possible to obtain other hybridomas also producing monoclonal antibodies directed against PCBLs. EXAMPLE 2: "DOUBLE SANDWICH ELIS A" PROTOCOL (for a 96-well ELI SA micro plate or any other support)

1) "Coating" with the 3C2 monoclonal antibody:

Dilution to 10 μg / ml in sterile phosphate buffered saline (PBS), 100 μl per well: 1 h at 37 ° C, with an adhesive cover, Wash 3 times with PBS-T een ^R 20 (0.05%).

2) Said with albumin (for example bovine serum):

1% dilution in sterile PBS 200 μl per well: 2 h at 37 ° C Wash 3 times. 3) Antiqene sol utions:

Dilution in PBS-Tween ^R 20 (0.05%) - SAB (1%)

- White: (8 in duplicate)

- Standard range: reference cancerous ascites diluted 1/15; 1/30; 1/60; ...; 1/1920 (each dilution in duplicate)

- Pathological or normal sera: dilutions 1/50 (32 sera in duplicate) 100 μl per well: 2 h at 37 ^C C Wash 4 times.

4) Biotin-labeled 3B1 monoclonal antibody: Dilution to 0.7 μg / ml in PBS-Tween * 20 (0.05%) - SAB

(0.5%)

100 μl per well: overnight (5 p.m.) at 4 ^C C

Wash 4 times.

5) Avidin * -peroxidase: in excess in sterile PBS-SAB (0.2%) 100 μl per well: 15 min. at 37 ° C Wash 4 times.

6) Substra t of peroxidase 100 μl per well: 20 min. at 20 ° C in the dark EXAMPLE 3: ASSAY OF PCBL 45-47 kDa IN THE SERUM BY THE RIA OR IRMA METHOD

The principle of the assay is substantially the same as for Example 2, but the detection of the PCBLs retained by the monoclonal antibody 3C2 is carried out by means of the radiolabelled monoclonal antibody 3B1, followed by counting.

This dosage applies to serums or other biological liquids, diluted beforehand.

Claims

RESELL ICA TIONS

1. Monoclonal antibodies directed against proteinase precursors, characterized in that: a) they are specifically directed against epitopes ⁵ of PCBL, giving rise to no immunological cross reaction with mature enzymes, cathepsin B-like, cathepsins B, H and L, and papain, b) they recognize epitopes on PCBLs which, - from one antibody to another, can differ.

2. Monoclonal antibodies according to claim 1, characterized in that:

- they belong to the class IgGi-k,

- they have a molecular weight of around 150kDa, - - they have a high affinity for PCBLs.

3. Anti-PCBL monoclonal antibodies, characterized in that they are capable of being obtained by a process comprising the steps: of immunization of an animal, according to the techniques

20 conventional, with purified PCBLs, fusion of antibody producing cells against PCBLs with immortal mouse myeloma cells, selection of hybrid cells capable of

To produce specific antibodies against PCBLs, and

- purification of the monoclonal antibodies produced.

4. Anti-PCBL monoclonal antibodies, characterized in that they are secreted by the clones

30 deposited at the CNCM under the numbers 1-890, 1-891 and 1-892 on July 17, 1989.

5. Hybridomas characterized in that they are capable of secreting a monoclonal antibody according to any one of claims 1 to 4. 35

6. Clones deposited at the CNCM under the numbers 1-890, 1-891 and 1-892 on July 17, 1989.

7. Process for the preparation of a hybridoma according to claim 5 or 6, by fusion according to

- usual techniques between mouse myeloma cells and antibody-producing cells from a mouse previously immunized with the PCBL antigen, followed by selection of those from hybrid cells which are capable of producing the

- anti-PCBL monoclonal antibodies.

8. Method according to claim 7, characterized in that the myeloma cells are SP2 / 0 myeloma cells originating from Balb / c mice and that the cells producing monoclonal antibodies

- are popliteal lymph node cells from Biozzi mice, good producers of antibodies.

9. Method for in vitro detection of the presence of PCBL in a biological sample, characterized in that it comprises:

- - bringing a biological sample from a patient likely to be affected by a disease accompanied by the secretion of PCBL into contact with a monoclonal antibody as defined above, under conditions suitable for producing a

²⁵ antigen-antibody complex, and

- detection of the possible immunological binding of the monoclonal antibody to the PCBLs of the cells of the biological sample.

10. Kit for the in vitro detection of PCBL of - 45-47 kDa in a biological sample, characterized in that it comprises:

35 a preparation of monoclonal antibody specific for PCBL of 45-47 kDa according to one of claims 1 to 4 serving as the first layer,

a preparation of another monoclonal antibody according to any one of claims 1 to 4 serving as a second layer, this monoclonal antibody reacting with another epitope of the form 45-47 kDa comprising a marker,

- a specific detection system for the labeled monoclonal antibody;

11. Composition of anti-PCBL monoclonal antibody, characterized in that it is intended for intravenous injection and comprises a monoclonal antibody according to any one of claims 1 to 4 carrying a radioactive marker, making it possible to locate by external scintigraphy or any other method, a malignant tumor or group of cells secreting PCBLs, after an immunological reaction between the labeled antibody and the PCBLs.

12. Immunotoxins characterized in that they consist of a monoclonal antibody according to any one of claims 1 to 4 associated with a toxin.