WO1992009699A1

WO1992009699A1 - Monoclonal antibody for use in diagnosing alzheimer's disease, hybridoma secreting said antibody and preparation method therefor

Info

Publication number: WO1992009699A1
Application number: PCT/BE1991/000083
Authority: WO
Inventors: Jean-Noël OCTAVE; Jean-Marie Maloteaux
Original assignee: L.F. Will & Cie (Belgique)
Priority date: 1990-11-27
Filing date: 1991-11-26
Publication date: 1992-06-11
Also published as: AU8866791A; BE1003316A5

Abstract

A monoclonal antibody directed against part of the extracellular domain of the APP precursor of peptide AβP is provided for recognising both kinds of lesions which are typical of Alzheimer's disease as well as the soluble form of the APP precursor. The use of this antibody for detecting said lesions and for assaying said soluble form of APP, as well as the hybridoma which produces said monoclonal antibody, a method for preparing said hybridoma, and a protein used as an antigen in said method, are also described.

Description

Monoclonal antibody useful for the diagnosis of Alzheimer's disease. secretory hybridoma of such a monoclonal antibody and process for its preparation.

The invention relates to a monoclonal antibody directed against a part of the extracellular domain of the precursor of the amyloid peptide, and its use for the diagnosis of Alzheimer's disease.

The invention also relates to the hybridoma capable of producing such an antibody, a process for the preparation of this hybridoma, as well as the antigenic protein used in this process.

Alzheimer's disease is responsible for more than half of the cases of senile dementia. According to some statistics, it is currently estimated that 10 to 15% of the population over the age of 65 and 20 to 40% of the population over the age of 80 have Alzheimer's disease.

Symptoms of Alzheimer's disease are memory impairment and a progressive decrease in intellectual capacity.

Alzheimer's disease is characterized by the loss of neurons and the presence of brain damage. These lesions consist on the one hand of extracellular deposits of amyloid substance present in the form of senile plaques and cerebrovascular deposits. On the other hand, there are neurofibrillary alterations which lead to the formation of intraneuronal lesions.

Many studies have been carried out to better understand the mechanism of formation of lesions characteristic of Alzheimer's disease.

From senile plaques, a peptide of 42 amino acids was isolated and called peptide A4 [C.L. Masters & Al., Proc. Natl. Acad. Sci. USA 82: 4245-4249 (1985)].

From the cerebrovascular deposits, a small peptide very close to the A4 peptide was isolated and called β peptide [G. Glenner & Al., Bioch. Bioph. Res. Com. 120: 885-890 (1984)]. Currently, we prefer to group these two names under the term ABP (Amyloid β Peptide).

Using a molecular probe (DNA probe _C ), the messenger RNAs corresponding to the precursor of the AϐP peptide were discovered and sequenced.

The precursor of the AβP peptide, a transmembrane glycoprotein, is called APP (Amyloid Peptide Precursor). So far 4 different versions of this precursor have been discovered. They respectively include 695 amino acids [J. Kang & Al., Nature 325, 733-736 (1987)], 751 amino acids [P. Ponte & Al., Nature 331, 525-527 (1988); R. Tanzi & Al., Nature 331, 528-530 (1988)], 770 amino acids [N. Kitaguchi & Al., Nature 331, 530-532 (1988)] and 563 amino acids [F. de Sauvage & Al., Science 245, 651-653 (1989)]. The precursor of 770 amino acids comprises a specific sequence of 55 amino acids described in patent application No. EP-A-0304013.

To facilitate understanding of the rest of the presentation, the complete sequence of the precursor of 770 amino acids, called APP 770, is given in FIG. 1, in the amino-carboxy direction.

The gene corresponding to the four precursors of the ABP peptide is located on chromosome 21.

The precursor APP is also present in soluble form in the cerebrospinal fluid of normal individuals and of patients suffering from Alzheimer's disease. This soluble protein comprises the extracellular domain of the precursor (N-terminal fragment) and has lost the transmembrane domain and the cytoplasmic domain (C-terminal fragment).

Indeed, antibodies directed against the precursor APP 695 recognized the soluble form of APP whereas antibodies directed against the cytoplasmic domain (C-terminal fragment of 43 amino acids) did not recognize the soluble form of APP [Weideman & Al., Cell. 57, 115-126 (1989)].

The neurofibrillary alterations, located inside the neurons, consist of helical filaments paired two by two in a characteristic way.

In reality, the diagnosis is not easy to establish, first because there are conditions that resemble Alzheimer's disease (vascular dementias, depressive syndromes in the elderly), then because there is no still a specific diagnostic test that is ante-mortem.

So far, different diagnostic paths have been opened:

the study of higher intellectual functions using neuropsychology techniques, represents a first diagnostic approach.

The cerebral scanner makes it possible to visualize the atrophy of the cerebral cortex and to eliminate certain other affections, in particular curable affections.

Nuclear Magnetic Resonance (NMR) as a new imaging technique allows better visualization of small vascular brain lesions and can improve the differential diagnosis between vascular dementia and Alzheimer's disease. These last two techniques actually carry out a diagnosis of exclusion.

The positron camera allows the study of the metabolism of the brain by analyzing the cerebral consumption of glucose. Different researches have shown that during Alzheimer's disease, glucose consumption is reduced, and have been able to locate the areas preferentially affected. Although providing valuable information, this technique does not yet constitute a real means of diagnosis.

Currently, the only sure diagnosis is the microscopic, post-mortem analysis of the brain tissue, showing the presence of deposits of amyloid substance. Previous research has shown that neurofibrillary changes are recognized by antibodies prepared against certain normal brain proteins, such as the MAP2 protein [K. Kosik & Al., Proc. Natl. Acad. Sci. USA 81, 7941-7945 (1984)] or the Tau protein [JP Brion & Al., Arch. Biol. (Brux.), 95, 229-235 (1985); K. Kosik & Al., Proc. Natl. Acad. Sci. USA 83, 4044-4048 (1986); A. Delacourte & Al., J. Neurol. Sci., 76, 173-186 (1986)].

On the other hand, these intracellular neurofibrillary alterations have so far never been recognized by antibodies prepared against the APP precursor. These only succeeded in recognizing the degenerate neurites which surround the amyloid deposit of the senile plaques.

The aim of the present invention is to prepare a monoclonal antibody which allows both post-mortem diagnosis of Alzheimer's disease, by highlighting both neurofibrillary changes and senile plaques, as well as ante-mortem diagnosis (dosage of the soluble form of APP in cerebrospinal fluid and detection of lesions characteristic of the disease in peripheral tissues, in particular in the olfactory mucosa).

The present invention relates to a monoclonal antibody directed against an amino acid sequence included in the extracellular part of the APP precursor.

This sequence is common to the transmembrane precursor as well as to its secreted soluble form. In the case of precursor 770, the extracellular part extends from the amino acids located at positions 1 to 699, the transmembrane part extends from the amino acids located at positions 700 to 723 and the cytoplasmic part extends from the amino acids located at positions 724 to 770 of FIG. 1.

The monoclonal antibody according to the invention is capable of recognizing the two types of lesions characteristic of Alzheimer's disease (senile plaques and neurofibrillary alterations) as well as the soluble form of the APP precursor present in serum and cerebrospinal fluid.

Preferably, said sequence is delimited by the amino acids located at positions 81 to 481 in FIG. 1. These amino acids correspond to amino acids 81 to 406 of the precursor APP 695, the complete sequence of which has been published by J. Kang et al. [Nature 325, page 734 (1987)].

In a preferred form of the invention, the monoclonal antibody is directed against a fusion protein between said amino acid sequence and β-galactosidase. This protein is obtained by expression of the corresponding DNA fragment _c , by E. cells. Coli transformed by the expression vector Puex I. The said DNA fragment _c is inserted into the Pst 1 site of this vector.

The synthesis of β-galactosidase is controlled by a powerful bacterial promoter which allows the expression in large quantity of said fusion protein.

The fusion protein thus obtained constitutes an antigen free from any contamination by other brain proteins which could be contained, for example, in neurofibrillary alterations.

In particular, the monoclonal antibody according to the invention belongs to the class of IgM and is produced by the hybridoma deposited at the ECACC on November 20, 1990 under the access number 90112001.

In particular, the recon monoclonal antibody masters an epitope included in the sequence delimited by amino acids 261 to 284 in FIG. 1.

The subject of the present invention is also the use of the above-described mci.oclonal antibody for the immunological detection of neurofibrillary alterations and senile plaques. This immunological test can in particular be carried out on biopsies of olfactory mucosa as well as on samples of brain tissue taken. post-mortem, in subjects with Alzheimer's disease.

In addition, the present invention relates to the use of this same antibody for the determination of the soluble form of the APP precursor present in the serum and the cerebrospinal fluid of patients suffering from Alzheimer's disease.

A subject of the present invention is also 1 * hybridoma producing the monoclonal antibody according to the invention, deposited with the ECACC on November 20, 1990 under the access number 90112001.

A subject of the invention is also a process for the preparation of the hybridoma according to which an animal is immunized, with a protein comprising an amino acid sequence included in the extracellular part of the APP precursor, a fusion is carried out between the spleen cells of this animal and the appropriate myeloma cells using a technique known per se (Milstein and Kohler, Nature 256 (1975)), the resulting hybrid cells are cloned and the cell clones which produce the desired monoclonal antibody are selected.

In a preferred form of the invention, said sequence is delimited by the amino acids located at positions 81 to 481 of the APP 770 precursor, such as sequence in FIG. 1.

According to an advantageous embodiment, the protein used to immunize the animal is a fusion protein obtained by expression of bacterial cells.

Advantageously, said cells are E. cells. coli transformed by a plasmid preferably resulting from the insertion into the Pst 1 site of the Puex I vector, of an amino acid sequence included in the extracellular part of the APP precursor. This sequence is preferably delimited by amino acids 81 to 481 of the precursor APP 770 of FIG. 1. Additional characteristics of the invention will become apparent during the detailed description of an example of the manufacture of the secretory hybridoma of the monoclonal antibody defined above.

1. Preparation of the antigen

To avoid contamination of the antigen by other proteins, the antigenic protein was not isolated from pathological tissues but was prepared by genetic expression, after transformation of bacterial cells with an adequate plasmid.

1.1 Preparation of the plasmid.

The restriction fragment Pst 1 coding for the amino acid sequence 81 to 481 of the precursor APP 770 was inserted into the Pst 1 site of the expression vector Puex I. The resulting plasmid coded for a fusion protein consisting of β- galactosidase followed by the extracellular sequence of the APP precursor (sequence 81 to 481 in Fig. 1).

1.2. Expression of the fusion protein.

E. cells Coli were transformed with this plasmid and cultured for 2 h at 42 ° C. This high temperature is necessary to induce expression because the plasmid contains the thermosensitive repressor CI 857.

1.3. Extraction and purification of the fusion protein.

The cells of E. Coli were collected by centrifugation at 2000 xg for 15 minutes. The pellet was resuspended in 20 ml of buffer containing 50 mM Tris-HCl pH8, 1 mM EDTA, 50 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and 8 mg of lysozyme. After 20 minutes of incubation of the suspension on ice, 4 ml of 1% deoxycholic acid was added. The solution was incubated at 37 ° C to achieve a high viscosity.

Bacterial DNA was digested by addition of

200 μl of DNAse at a concentration of 1 mg / ml and MgCl ₂ until reaching a final concentration of 10 mM.

After 15 minutes of incubation at room temperature, the inclusion bodies were collected by centrifugation at 5000 g for 5 minutes, then washed three times in the buffer described above supplemented with 0.5% Triton X-100.

After preparative electrophoresis on acrylamide gel and staining of the gel with 0.1% Coomassie blue, the band corresponding to the fusion protein was excised. This excised gel fragment was homogenized in a volume of physiological saline solution, buffered with phosphate. The homogenate was incubated overnight to allow diffusion of the protein, and centrifuged at 12,000 xg for 5 minutes.

The fusion protein is then collected in the supernatant.

2. Immunization.

50 μg of the fusion protein diluted in complete Freund's adjuvant were injected intraperitoneally into Balb / C mice on days 0, 7, 18, 28 and 35. 42 days after the last inoculation, an immune response was observed in the serum of mice.

3. Preparation of hybridomas.

Spleen cells from immunized mice were isolated in 10 ml of Gibco DMEM culture medium supplemented with 100 U / ml of penicillin and 100 U / ml of streptomycin.

The solution is centrifuged at 1200 g for 10 minutes and the pellet is washed 2 times with this same culture medium. The splenic cells thus harvested were stained with trypan blue and counted in a cell.

BurJcer.

The spleen cells and myeloma cells (NSO) were mixed in a 5/1 ratio. The cell mixture was centrifuged at 2500 g for 5 minutes and incubated in the presence of 1 ml of polyethylene glycol 1500 (PEG) at a concentration of 0.5 g / 1500 ml of 75 mM HEPES buffer.

After progressive dilution in the DMEM culture medium described above, the cells were collected by centrifugation at 1200 g for 10 minutes and then resuspended in the HAT selection medium from Gibco. This medium only allows the growth of hybrid cells.

The suspension was distributed in plates with 96 wells at the rate of 200 μl of suspension per well. The cells were kept in culture under a humidified atmosphere containing 5% CO ₂ .

After 10 days of culture the supernatants of the wells contained sufficiently developed hybrid colonies.

4. Selection of the hybridoma producing the desired monoclonal antibody.

The culture supernatants of the hybrid cells were tested for the production of the desired monoclonal antibody by immunoenzymatic reaction (ELISA).

The reference antigen (the preparation of which is described in point 1) at a concentration of 0.5 μg / ml in a buffer containing 50 mM glycine of pH 9.2 was fixed on a solid support (for example a microtiter plate).

The antigen was then incubated at 37 ° C for 2 h with 50 μl of the supernatant from hybridoma cultures.

The monoclonal antibody capable of recognizing the reference antigen is retained by the fixed antigen and

“Its presence was detected using a rabbit anti-mouse IgG anti-serum, this anti-serum being demonstrated by protein A labeled with peroxidase.

For this, after incubation of the wells with the supernatant, they are incubated at room temperature for 1. h with 50 μl of the antiserum then with protein A. After each incubation, the plates are washed 5 times with a 0.25 mM NaCl solution containing 0.2% of Tween 20.

The enzymatic activity of peroxidase was developed using 0.04% of ortho-phenylenediamine in H ₂ O ₂ at 0.006% and then stopped by adding 25 μl per cup of H ₂ SO ₄ 4M.

Reading the absorbance at 425 and 620 nm made it possible to locate the hybridoma producing the monoclonal antibody recognized by the reference antigen.

5. Production of the desired monoclonal antibody.

The desired monoclonal antibody is produced by intraperitoneal injection into mice of the appropriate hybridoma (selected in point 4).

The ascites fluid taken from these mice contains the desired monoclonal antibody at a high concentration.

6. Characterization of the monoclonal antibody.

The immunoblotting technique made it possible to study the specificity of the monoclonal antibody, firstly, in relation to an antigen obtained by genetic expression and secondly in relation to the antigen isolated from brain tissues.

6.1. Characterization of the monoclonal antibody compared to an antigen obtained by genetic expression.

COS cells (monkey renal cell line) were transfected with the transfection vector pKCR 3 into which the DNA fragment _c coding for the precursor APP 770 had been previously introduced. The COS cells were chosen for their capacity to express a glycosilated protein, that is to say very close to the native proteins.

These COS cells were cultured in DMEM medium from Gibco containing 5% fetal calf serum, then incubated for 24 h in culture medium without serum. These cells, as well as their culture medium, were then analyzed jointly by the immunoblot technique.

In summary, the cells to be analyzed were lysed in a reducing buffer containing SDS. On the other hand, the proteins coming from the culture medium without serum were desalted by chromatography on a column of Sephadex G25, then concentrated by lyophilization and finally solubilized in the same buffer as the COS cells.

The proteins associated with the COS cells and the proteins originating from the culture medium were then subjected to electrophoresis on polyacrylamide gel under denaturing conditions.

The proteins were transferred from the gel to a nitrocellulose membrane using a semi-dry transfer device supplied by Biometra.

To avoid non-specific protein binding, the membrane was incubated in a solution containing 5% skimmed milk before being incubated overnight with ascites fluid, diluted to 1/1000, containing the monoclonal antibody obtained at point 5.

The antigen-antibody reaction is detected by incubation of the membrane with a second biotinylated anti-mouse antibody produced in goats and then with the streptodivine-alkaline phosphatase complex.

For the analysis of the proteins associated with the transfected COS cells and the analysis of the proteins originating from the culture medium, major bands corresponding to proteins of approximately 130 kDa are obtained in the gel. These different bands correspond to the APP precursors.

Analysis of untransfected COS cells shows weak bands in the gel, corresponding to a low level of endogenous expression of the APP protein by COS cells.

The results of this analysis therefore show that the monoclonal antibody according to the invention specifically recognized the APP protein expressed by COS cells transfected with the DNA fragment _c coding for APP 770. 6.2. Characterization of the monoclonal antibody compared to an antigen contained in brain tissue.

Brain tissue taken from rats and humans was analyzed by the immunoblot technique using the monoclonal antibody according to the invention.

Whole rat brain and human frontal cortex samples were homogenized (1 g / ml) in 0.1 M MES buffer pH 6.8 containing 1 mM EDTA, leupeptin (25 μg / ml ), trypsin inhibitor (100 μg / ml) and phenylmethylsulfonyl fluoride (0.5 mM).

The homogenates were centrifuged at 90,000 g for 60 minutes at 4 ° C and the supernatant was retained for the immunoblot experiments.

The proteins of the supernatant were analyzed by immunoblotting in the same way as the COS cells (see point 6.1).

The results of the analysis show bands at PM between 100 and 135 kDa. These bands represent the different versions of the APP precursor which have been recognized by the monoclonal antibody according to the invention.

Bands at lower PM were also detected by the antibody.

6.3. Characterization of the epitope recognized by the antibody.

The restriction fragment Pst I coding for the amino acid sequence 81 to 481 of the precursor APP 770

(Fig. 1), originally used to produce the antigen, was divided into seven different fragments which were amplified by the polymerase chain reaction described by Saiki in 1988 (Saiki RK et al., Science 239, 487- 491,

1988). These different fragments were cloned into the bacterial expression vector pUEX II, and the corresponding proteins, expressed in the form of fusion proteins with β-galactosidase, were analyzed after polyacrylamide gel electrophoresis, transfer to nitrocellulose membrane, and immunodetection using the antibody according to the invention. The recognition of certain protein fragments made it possible to define the epitope of the antibody, within a sequence of 24 amino acids delimited by amino acids 261 to 284 of FIG. 1 (This sequence is underlined in Fig. 1).

7. Use of the monoclonal antibody as a diagnostic tool for Alzheimer's disease.

The monoclonal antibody according to the invention was used for the immunohistochemical analysis of sections of normal human brain and of the brain of patients suffering from Alzheimer's disease. The analysis was carried out according to the peroxidase-antiperoxidase process.

Brain tissue samples from the hippocampus and temporal cortex were taken post-mortem, fixed in 10% formalin and coated with paraffin. 10 μm thick sections were made.

After removing the paraffin and rehydrating the sections, the activity of the endogenous peroxidase was blocked by incubation for 30 minutes in a solution of H ₂ O ₂ in 70% methanol. The tissue sections were then incubated for 1 h in normal goat serum diluted 10 times in a saline physiological solution buffered with 0.05 M Tris (TBS) (pH 7.4).

The sections were then incubated for 16 h at room temperature with the ascites fluid containing the monoclonal antibody according to the invention, diluted to ^1/250 in TBS containing 1.3 g / l of sodium azide and 10% normal goat serum. The sections previously washed in TBS are then successively incubated for 30 minutes with anti-mouse antibodies produced in goats, then for 30 minutes with the peroxidase-antiperoxidase complex.

0.03% diaminobenzidine in Tris HC10.05 pH 7.6 with 0.015% H ₂ O ₂ reveals the reaction of the antibody according to the invention with the antigen contained in the brain tissues analyzed.

The results show no coloration of sections of normal human brain by the antibody according to the invention, whereas the latter colors both the neurofibrillary alterations and the senile plaques found in pathological brain tissues.

The marking of the neurofibrillary alterations was confirmed by the observation under the electron microscope of tissue sections incubated with the monoclonal antibody according to the invention.

Claims

1.- Monoclonal antibody directed against an amino acid sequence included in the extracellular part of the APP precursor, capable of demonstrating the two types of lesions characteristic of Alzheimer's disease and the soluble form of the APP precursor.

2.- monoclonal antibody according to claim 1, characterized in that said sequence is delimited by the amino acids located at positions 81 to

481 of FIG. 1.

3.- Monoclonal antibody according to any one of the preceding claims, produced by the hybridoma deposited at the ECACC under the access number 90112001.

4. Monoclonal antibody according to any one of the preceding claims, characterized in that it recognizes an epitope included in the amino acid sequence delimited by amino acids 261 to 284 of FIG. 1.

5. Use of the monoclonal antibody according to any one of the preceding claims, for the immunological detection of neurofibrillary alterations and of the senile plaques characteristic of Alzheimer's disease.

6.- Use according to claim 5, characterized in that said detection is carried out, ante-mortem, on samples of the olfactory mucosa.

7. Use according to claim 5, characterized in that said detection is carried out, post-mortem, on samples of brain tissue.

8. - Use of the monoclonal antibody according to any one of claims 1 to 4 for the determination of the soluble form of the APP precursor present in the serum and the cerebrospinal fluid of patients suffering from Alzheimer's disease .

9.- Hybridoma producing a monoclonal antibody according to any one of claims 1 to 4.

10.- Hybridoma according to claim 9, deposited with the ECACC under the access number n ° 90112001.

11. A process for the preparation of the hybridoma according to any one of claims 9 and 10, characterized in that:

1) an animal is immunized with a protein comprising an amino acid sequence included in the extracellular part of the APP precursor;

2) according to a known method, a fusion is carried out between the spleen cells of this animal and appropriate myeloma cells, the cells resulting from said fusion are cloned and the cell clones which are found to produce the desired monoclonal antibodies are selected.

12.- Method according to claim 12, characterized in that said sequence is delimited by amino acids 81 to 481 of FIG. 1.

13.- Method according to any one of claims 11 and 12, characterized in that said protein is a fusion protein obtained by expression of bacterial cells.

14.- Method according to claim 13, characterized in that said cells are E cells. coli transformed by a plasmid.

15.- Method according to claim 14, characterized in that said plasmid results from the insertion into the Pst 1 site of the vector Puex I, of an amino acid sequence included in the extracellular part of the APP precursor.

16.- Method according to claim 15, characterized in that said sequence is delimited by amino acids 81 to 481 of FIG. 1.

17.- Protein used in the process according to any one of claims 11 to 16, to immunize the animal.