JP2006267124A - Method for examining wt1-related disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for easily and reliably detecting the course of cure and a prognosis of a WT1-related disease such as leukemia, a solid cancer, or an atypia. <P>SOLUTION: The method is an examination method of determining the presence of the WT1-related disease in a subject, and the course of cure and the prognosis thereof, and the method measures an antibody amount against a WT1 in a specimen and uses a measured value and a change with the lapse of time, as a clinical index in the examination. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a test method for determining the presence or treatment course and prognosis of a WT1-related disease.

  The WT1 gene is a zinc finger type transcription factor isolated as a causative gene for Wilms tumor, and its gene product (WT1 protein) consists of a suppression domein and an activation region ( activation domein) and zinc finger structure.

  The present inventors can detect MRD (residual leukemia cells) of acute leukemia by measuring that the expression level of the WT1 gene is high in acute leukemia, and the expression level is inversely correlated with prognosis. (Blood, Vol.84, No.9, p3071 (1994)). The present inventors have also found that various solid cancers and variants can be detected by measuring the expression level of the WT1 gene (WO97 / 39354).

  Therefore, the measurement of the expression level of the WT1 gene, which consists of measuring the transcript or translation of the WT1 gene, has clinically significant significance for these WT1-related diseases.

  An object of the present invention is to provide a new technique effective for diagnosis of WT1-related diseases, therapeutic effects, prognosis determination, and the like.

  In the course of continuing research for the purpose described above, the present inventors detect antibodies against WT1 in specimens such as sera of patients with WT1-related diseases, and the amount of the WT1 antibody decreases as the disease cures. In other words, the present inventors have found that the amount of the WT1 antibody becomes a new clinical index of WT1-related diseases, and the measurement itself can determine the existence of the WT1-related diseases, the course of treatment and the prognosis with high reliability. The present invention has been completed based on this knowledge.

  The present invention is a test method for determining the presence or treatment course and prognosis of a WT1-related disease in a subject, measuring the amount of antibody against WT1 in a sample, and using the measured value as a clinical index in the test A method for testing a WT1-related disease is provided.

  In particular, the present invention provides the above test method wherein the amount of antibody against WT1 is measured by an immune reaction using the WT1 antigen; the test wherein the WT1 antigen is a WT1 protein that contains a suppressor region and an activation region and lacks a zinc finger Method: The above test method wherein the antibody against WT1 is an IgG antibody or an IgM antibody; the test method using a change in the amount of antibody against WT1 over time as a clinical index; the above ratio using a quantitative ratio of IgG antibody to IgM antibody against WT1 as a clinical index Provided are the above-described inspection method for determining progression from myelodysplastic syndrome to leukemia; and the above-described inspection method for determining complete remission of leukemia.

  Hereinafter, indications with abbreviations such as amino acids, peptides, base sequences, nucleic acids, etc. in this specification are the provisions of IUPAC and IUB, “Guidelines for the creation of specifications including base sequences or amino acid sequences” (edited by the JPO) And follow the convention symbols in the field.

  The WT1-related disease testing method according to the present invention is carried out by measuring the amount of antibody against WT1 in a specimen.

  In the above, examples of specimens (samples) are specimens derived from patients with WT1-related diseases or patients after treatment thereof, and are generally known to have antibodies, for example, various body fluids such as blood and urine, without limitation. can do.

  Examples of WT1-related diseases include various diseases known as WT1-related diseases such as leukemia, solid cancer, and atypia. It also includes WT1-related diseases found in the future.

The antibody against WT1 measured by the present invention refers to an antibody against the expression product (WT1 protein) of the WT1 gene [see Cell, 60 , 509 (1990); Natute, 343 , 774 (1990)]. Includes all of the antibodies found in the subject. Such antibodies include various immunoglobulins such as IgG antibodies, IgA antibodies, and IgM antibodies.

  Measurement of the amount of antibody against WT1 according to the method of the present invention can be carried out satisfactorily according to various methods commonly used in antibody measurement techniques, for example, immunoassay using WT1 antigen. As a specific example of this immunoassay, for example, a solid phase sandwich method can be exemplified.

  This solid phase sandwich method is performed, for example, as follows. That is, first, an antigen (WT1 antigen) capable of specifically reacting with an antibody against WT1 (WT1 antibody) to be measured is immobilized on a solid phase, and a specimen as a test sample is added thereto. As a result, an antigen-antibody reaction occurs between the immobilized antigen and the antibody in the sample, and the WT1 antibody present in the specimen binds to the immobilized antigen. Next, the bound antibody is detected using an antibody detection reagent, and the WT1 antibody present in the test sample is measured.

  In the above, the antibody detection reagent is solid-phased, thereby capturing the antibody in the specimen, and then adding the WT1 antigen to bind to the WT1 antibody in the captured antibody, and further labeling the specific antibody of the antigen By binding, the target WT1 antibody present in the test sample can also be detected and measured.

  Selection of various means in these measurement methods, modification thereof and the like are all well known to those skilled in the art, and there is no particular limitation in the present invention, and any of these methods can be adopted [ See “Procedures for clinical testing”, Kanehara Publishing, 1995, etc.].

  The antibody detection reagent for detecting various immunoglobulins such as IgG is not particularly limited, and various commonly used reagents can be used. For example, use preparations consisting of anti-human IgG antibodies, anti-human IgM antibodies, anti-human Ig (G + M) antibodies that specifically bind to human IgG, human IgM (or both), etc. for which measurement is desired it can. These can be obtained as commercial products and can also be prepared according to conventional methods.

  The WT1 antigen is not particularly limited as long as it is an antigen specific to the WT1 gene product, and can be employed in the present invention. Such an antigen can be the WT1 protein itself, or at least a fragment thereof having an antigenic determinant specific to the WT1 protein. Examples of these WT1 antigens include those chemically synthesized based on the amino acid sequence information of the WT1 protein and those prepared by genetic engineering techniques without limitation.

  Whether or not the WT1 antigen to be employed can be used favorably in the present invention is determined by, for example, reacting with the antibody using an antibody already confirmed as a WT1 antibody possessed by a WT1-related disease patient. It can be easily confirmed by testing according to conventional methods.

  In the present invention, a WT1 antigen particularly suitable as an antigen for the above measurement system includes a WT1 protein suppression region and activation region and lacks a zinc finger. Among them, a protein having a partial sequence consisting of the 1-294 amino acid sequence in the amino acid sequence of the WT1 protein (hereinafter referred to as “HWT3”) is more preferable. The HWT3 can be produced by a genetic engineering technique using a known WT1 gene, for example, by a method described in Examples described later as specific examples and a method analogous thereto.

  The production of the WT1 antigen by genetic engineering techniques using the WT1 gene can be performed according to a conventionally known general gene recombination technique. More specifically, a transformant is prepared by preparing a recombinant DNA capable of expressing a desired WT1 gene in a host cell, introducing this into a host cell, transforming it, and culturing the transformant. The desired WT1 antigen can be produced as an expression product inside or outside the cells.

Various operations that can be employed here, such as chemical synthesis of some genes, cleavage, deletion, enzyme treatment for the purpose of addition or binding, isolation, purification, selection, etc. Cultures of transformed cells can all follow conventional methods (for example, “Molecular Genetics Experimental Method”, published by Kyoritsu Publishing Co., Ltd. 1993; “PCR Technology”, published by Takara Shuzo Co., Ltd. 1990, Science, 224 , 1431) (1984); Biochem. Biophys. Res. Comm., 130 , 692 (1985); Proc. Natl. Acad. Sci., USA., 80 , 5990 (1983); Moplecular Cloning, by T. Maniatis et al., See Cold Spring Harbor Laboratory (1982)).

  If desired, the WT1 antigen can be separated from each other using various physical and chemical properties (for example, “Biochemical Data Book II”, pages 1175-1259, first edition, first edition, June 23, 1980). And the like, and the like can be separated and purified from the above expression product.

  In measuring the amount of WT1 antibody in a sample in the test method for WT1-related diseases according to the present invention, known techniques, means, measurement reagents used for them, and the like can be suitably used.

  For example, when the solid phase method is employed in the above measurement method, the antigen or antibody of the measurement system is immobilized on the solid phase according to a conventional method. As the solid phase, commonly used insoluble and inert carriers can be widely used. For example, glass, cellulose powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethylcellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methylvinylether-maleic acid Examples include sticks, beads, microplates, test tubes and the like made of various materials such as a polymer, an amino acid copolymer, and an ethylene-maleic acid copolymer.

  The method for immobilizing the antigen or antibody is not particularly limited, and either physical binding or chemical binding can be used. Typically, for example, as a covalent bond method, diazo method, peptide method (acid amide derivative method, carboxyl chloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide activated polysaccharide method, Cellulose carbonate derivative method, method using a condensation reagent, etc.), alkylation method, carrier binding method using a crosslinking reagent (using glutaraldehyde, hexamethylene isocyanate, etc. as a crosslinking reagent), carrier binding method using Ugi reaction, etc. A method utilizing a chemical reaction: an ion binding method using a carrier such as an ion exchange resin: a physical adsorption method using a porous glass such as a glass bead as a carrier.

  The labeling agent in each measurement system is not particularly limited, and any conventionally known one or one that can be used in the future can be used. Specific examples include various radioisotopes commonly used in immunoassays, enzymes such as alkaline phosphatase (ALP), peroxidase (POX), fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (RITC), etc. Fluorescent substances, other examples such as 1N- (2,2,6,6-tetramethyl-1-oxyl-4-piperidyl) -5N- (aspartate) -2,4-dinitrobenzene (TOPA) without limitation it can.

  Examples of the enzyme labeling substance for enzyme labeling include microperoxidase, chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, D- Illustrative examples include nuclease and P-nase. Labeling methods using these labeling substances can be carried out according to known methods (for example, “monoclonal antibody”, Ikuzaki Ikuo et al., Kodansha Scientific, 1984; “Enzyme Immunoassay” 2nd edition, Eiji Ishikawa et al. Author, medical school, 1982).

  The enzyme activity can also be measured according to a known method according to the type of enzyme used. For example, when peroxidase is used as the labeling enzyme, ABTSJ (2,2′-azino-bi (3′-ethylbenzthiazolinesulfonic acid)) is used as the substrate, and when alkaline phosphatase is used, p- Nitrophenyl phosphate can be used to measure the degradation of each substrate using a spectrophotometer, etc. ("Enzyme immunoassay", 2nd edition, Eiji Ishikawa et al., Medical Shoin, 1982, etc.) reference).

  In addition, also when using the labeled body by a radioisotope, a fluorescent substance, etc. instead of the said enzyme label | marker, the measurement of the said labeled body can be implemented according to a well-known method.

  The solvent used in the measurement system may be any of those commonly used as long as it does not adversely affect the reaction. Specifically, a citrate buffer solution, a phosphate buffer solution, a Tris salt buffer solution. A buffer solution having a pH of about 5-9, such as an acetate buffer, can be suitably used.

  The immune reaction (binding) conditions are not particularly limited, and usual conditions generally used in this type of measurement method are employed. In general, the reaction can be carried out under a temperature condition of 45 ° C. or less, preferably about 4-40 ° C., taking about 1-40 hours.

  The greatest feature of the present invention is that the WT1 antibody amount in the specimen thus measured is used as a clinical index for determining the presence or treatment course and prognosis of a WT1-related disease. That is, the amount of WT1 antibody in a WT1-related disease patient is significantly increased as compared with that in healthy individuals, and decreases with the treatment of the patient. Therefore, in the present invention, it is possible to determine the presence or treatment course and prognosis of a WT1-related disease using the amount of WT1 antibody, preferably the change over time as a clinical index, and the amount of WT1 antibody in a sample can be determined. The decrease can be grasped as a desirable clinical index in the determination.

  In particular, the present inventors have found that the amount of IgM-type WT1 antibody and IgG-type WT1 antibody, changes with time, and changes in the ratio of both are more effective in determining the presence or treatment course and prognosis of WT1-related diseases. Has been found to be a good indicator.

  In addition, in WT1-related diseases, particularly in patients with myelodysplastic syndromes (MDS), it has been confirmed that the class switching of the WT1 antibody from IgM to IgG occurs in relation to the progress of the disease. Therefore, by examining the time-dependent change in the abundance ratio of the above-mentioned IgM type WT1 antibody and IgG type WT1 antibody, MDS is refractory anemia (RA, refractory anemia) to refractory anemia (RAEB, RA with It is possible to confirm that it progresses to RAEB (RAEB-t, RAEB in transformation) accompanied by deformation through excess of blasts), and eventually leukemia.

  Furthermore, the present inventors can confirm the maintenance of the complete remission state by examining the disappearance of the WT1 antibody over time when the WT1-related disease such as leukemia is in complete remission. Heading.

  In addition, detection of WT1 antibody in a WT1-related disease patient, that is, confirmation of a WT1 antibody-positive patient means confirmation that the patient has a humoral immune response against WT1. Therefore, detection of the WT1 antibody itself is useful in determining or diagnosing the immune response ability of the patient in a WT1-related disease. Because patients with an immune response against WT1 have a lower immune response than patients who have not, WT1 may have a worse prognosis. is important. Thus, measurement of WT1 antibody in WT1-related disease patients is extremely useful for diagnosing WT1-related diseases, particularly immune response ability (to cancer) in various cancer patients.

  In carrying out the test method of the present invention, it is convenient to use a test agent for the test, preferably a test kit. Therefore, the present invention also provides a test agent suitable for a test method for determining the presence or treatment course and prognosis of such WT1-related diseases.

Such a test agent of the present invention can contain, as an active ingredient, a WT1 antigen, that is, an antigen reagent that undergoes an antigen-antibody reaction with the WT1 antibody to be measured. The test agent can further contain any reagent such as an antibody detection reagent used in the measurement system, and suitable reagents for the convenience of the measurement, such as an antibody diluent, reaction, etc. Diluents, buffers, washings, labeled body detection reagents and the like may be included.
[Example]

  Hereinafter, the contents of the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these.

Measurement of WT1 antibody
(1) Production of WT1 antigen The WT1 gene (Blood, Vol.91, p.2969 (1998)) is incorporated into the plasmid pBluescriptII (Stratagene), and this pBluescriptII / WT1 (+ / +) is used as a template for 5 ' PCR was performed using a primer added with an EcoRI recognition sequence (SEQ ID NO: 1) and a primer added with a Not I recognition sequence at the 3 ′ end (SEQ ID NO: 2). The WT1 cDNA cDNA fragment encoding the amino acid sequence of was amplified. This DNA fragment was then incorporated into pGEX-5X-3 (Amersham Pharmacia Biotech) and transfected into E. coli BL21 (DE3). In order to increase the solubility of the fusion protein, the Thioredoxin expression plasmid pT-Trx was co-transfected. After culturing this E. coli overnight, the E. coli solution was diluted 10-fold, cultured at 37 ° C. for 1.5 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM, and further cultured for 5 hours. After collecting the cells, add lysate (50 mM Tris / HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM Prefabloc SC (Boehringer Mannheim), 10 μg / ml leupeptin, 1 mM DTT) and crush with ultrasound. The supernatant was recovered after centrifugation. The fusion protein in the supernatant was bound to glutathione sepharose 4B (Amersham Pharmacia Biotech), washed, and the fusion protein was eluted with an elution buffer (50 mM Tris · HCl (pH 7.5), 150 mM NaCl, 20 mM reduced glutathione,
GST-WT1 fusion protein HWT3 was recovered by elution with 1 mM DTT).

  Similarly, HWT2 (WT1 cDNA fragment encoding amino acid sequence 1-181) and HWT4 (WT1 cDNA fragment encoding amino acid sequences 182-294) were produced as follows.

  That is, using pBluescriptII / WT1 (+ / +) as a template, a primer containing an EcoRI recognition sequence at the 5 ′ end and a primer containing a Not I recognition sequence at the 3 ′ end (the sequence shown in SEQ ID NO: 1 for HWT2) 5 ′ plummer and 3 ′ primer of the sequence shown in SEQ ID NO: 3, and for HWT4, 5 ′ primer of the sequence shown in SEQ ID NO: 4 and 3 ′ primer of the sequence shown in SEQ ID NO: 2, respectively. PCR was performed to amplify the desired DNA fragment, each DNA fragment was incorporated into pGEX-5X-3 (Amersham Pharmacia Biotech) and transfected into E. coli BL21 (DE3). From this E. coli, WT1 fusion proteins HWT2 and HWT4 were recovered in the same manner as described above.

  The structure of each protein of HWT2, HWT3 and HWT4 obtained according to the above is schematically shown in FIG. 1 in comparison with WT1.

In FIG. 1, 1-181 is a suppression region, 182-294 region is an activation region, followed by a zinc finger.
(2) Reactivity test between WT1 antigen and WT1 antibody The reactivity between the WT1 antigen prepared in (1) above and the following WT1 antibody was tested as follows.

That is, GST fusion protein was dissolved in PBS at a concentration of 250 μg / ml and adsorbed on a nitrocellulose membrane (Optitran, Shleicher & Schuell) at a concentration of 25 μg / cm ² . This nitrocellulose membrane was washed with PBS, immersed in a 2% bovine serum albumin (BSA) solution for 2 hours, and then mounted on a dot-blot apparatus (Scheicher & Schuell). 20 μl of WT1 antibody solution was placed on a nitrocellulose membrane adsorbed with GST fusion protein, reacted for 1 hour, washed with PBS, and further reacted with HRP-conjugated anti-IgG antibody for 1 hour. After washing, the membrane is immersed in a substrate solution (Renaissance (registered trademark), NEN Life Science Products) and allowed to react. After drying, the membrane is overlaid on a photosensitive film (Hyperfilm MP, Amersham Life Science) to expose the film. The density of the upper band was measured using a computerizable scanning analysis system (Molecular Dynamics), and the antibody titer was calculated as Densitometric Units.

The WT1 antibody used is as follows.
S-Cruz180: Rabbit polyclonal antibody against GST fusion protein containing the N-terminal 180 amino acid sequence of human WT1 (manufactured by Santa Cruz Biotechnology, Inc.)
Pharmingen: a mouse anti-human WT1 protein monoclonal antibody (Pharmingen) that recognizes the exon 5 sequence, obtained by immunization with a synthetic peptide corresponding to the exon 5 sequence of human WT1
(3) Results The results are shown in FIG.

  Figure 2 shows the following. That is, it was revealed that HWT2 reacts only with rabbit polyclonal antibody S-Cruz180, while HWT4 reacts only with mouse monoclonal antibody Pharmingen, whereas HWT3 reacts with both.

  Hereinafter, HWT3 was used as an antigen for detecting the WT1 antibody in order to widely detect the WT1 antibody in the specimen.

WT1-related disease test 1
(1) Preparation of specimen The test serum was diluted 2500 times with PBS containing 2% BSA and 0.05% Tween 20, and used as a specimen sample.
(2) Adjustment of antigen adsorption membrane
The HWT3 fusion protein solution (250 μg / ml) was adsorbed on a nitrocellulose membrane (Optitran, Shleicher & Schuell) at a concentration of 25 μg / cm ² for 1 hour. This membrane was washed with PBS, immersed in a blocking solution (2% bovine serum albumin solution) for 2 hours, washed and used as an antigen-adsorbing membrane.
(3) Measurement of WT1 antibody Using the antigen-adsorbing membrane obtained in (2) above, each of the WT1 antibodies (IgG and IgM) contained in the specimen sample prepared in (1) was measured according to (2) of Example 1. It was measured.

That is, a nitrocellulose membrane adsorbing GST fusion protein was attached to a dot-blot apparatus, and 20 μl of a test sample (diluted serum) was placed on this membrane and incubated for 1 hour. After washing with PBS, the membrane in 1% BSA / PBS is HRP-conjugated goat anti-human IgM antibody (ICN
Pharmaceuticals Inc.) and in the case of IgG antibody, an HRP-conjugated rabbit anti-human IgG antibody (ICN Pharmaceuticals Inc.) was further reacted for 1 hour. After washing with PBS, the membrane is immersed in the substrate solution to react, dried, the membrane is overlaid on the photosensitive film to expose the film, the density of the band on the film is measured, and the antibody titer (Densitometric Units) is determined. Asked. These operations were all performed at room temperature.
(4) Measurement results
(a) 43 healthy adults and 33 patients with WT1-related disease (12 myelodysplastic syndromes (MDS), 12 patients with acute myeloid leukemia (AML), 4 patients with acute lymphoblastic leukemia (ALL) and chronic myeloid The results obtained according to the above method using each specimen of leukemia (CML) patients are shown in FIGS.

  In each figure, the vertical axis indicates antibody titer (Densitometric Units), and the horizontal axis indicates each specimen.

  From the results shown in FIG. 3, it can be seen that patients with WT1-related diseases have IgG-type WT1 antibodies that are significantly higher (p <0.001) than healthy individuals.

  From the results shown in FIG. 4, it can be seen that AML and MDS patients have significantly higher IgG type WT1 antibodies than healthy individuals.

  From the results shown in FIG. 5, it can be seen that patients with WT1-related diseases have IgM-type WT1 antibodies that are significantly higher (p = 0.003) than healthy individuals.

  From the results shown in FIG. 6, it can be seen that AML and MDS patients have significantly higher IgM type WT1 antibodies than healthy individuals.

  From these, it is clear that the presence diagnosis of WT1-related diseases such as AML, ALL, CML, MDS and the like can be performed by measuring IgG type and / or IgM type WT1 antibodies.

Examination of prognosis of patients with WT1-related diseases In the same manner as in Example 2, IgG type WT1 antibody titers were measured before and after treatment (during complete remission (CR)) in six patients with acute leukemia.

  The obtained results are shown in FIG.

  In FIG. 7, the horizontal axis represents the pretreatment and complete remission of each patient, and the vertical axis represents the WT1 antibody titer of each specimen. Each line (1) to (6) in the figure represents a patient with WT1-related disease, and the solid line in the figure is 4 patients who achieved complete remission by peripheral blood stem cell transplantation, and the wavy line is complete remission by chemotherapy There are 2 patients who became.

  The following can be seen from FIG. That is, it is clear that the IgG type WT1 antibody titer detected before the treatment by the method of the present invention falls below the detection sensitivity when the treatment is effective and enters complete remission (CR).

  From the above, it became clear that the effectiveness of treatment can be confirmed and diagnosed by following the IgG type WT1 antibody titer, in other words, the above antibody titer reflects the amount of leukemia remaining.

WT1-related disease test 2
(1) Preparation of specimens 43 healthy adults and 46 WT1-related disease patients (16 myelodysplastic syndromes (MDS), 16 acute myeloid leukemia (AML) patients, 7 acute lymphoblastic leukemia (ALL) patients and Serum was collected from 7 patients with chronic myelogenous leukemia (CML). The MDS patients are 6 refractory anemia (RA) patients, 4 refractory anemia with excess blasts (RAEB) patients and 6 RAEB (RAEB-t) patients with deformity.

The test serum was stored at −20 ° C. until use.
(2) Preparation of WT1 antigen for measurement of WT1 antibody
A DNA sequence corresponding to the WT1 partial protein (1-294 (HWT3)) was amplified by the PCR method in the same manner as in (1) of Example 1, and this was amplified into a plasmid vector pET- containing the C-terminal His-Tag sequence. It was cloned into 21b (+) (Novagen Inc., Madison, Wis.). The resulting plasmid was transfected into E. coli XL1-Blue and the desired transformants were examined by restriction enzyme mapping and DNA sequencing. Plasmid DNA was then transfected into E. coli BL21 (DE3) (Stratagene, La Jolla, CA) to prepare recombinants.

Recombinant E. coli BL21 (DE3) was cultured at 37 ° C. until A600 = 0.6, and incubated for 4 hours in the presence of 0.1 μM IPTG to induce the WT1 partial protein. E. coli was recovered by centrifugation at 6000 g for 10 minutes, resuspended in 4 ml of buffer A (100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, pH 8.0) (per 200 ml of culture medium), and stored at −80 ° C. . After thawing on ice, E. coli was sonicated (3 times, 2 minutes) and centrifuged at 6000 g for 10 minutes. The pellet containing inclusion bodies is resuspended in buffer B (100 mM NaH ₂ PO ₄ , 10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 6 M urea, 15 mM imidazole, 20 mM β-ME) and gently stirred on ice For 1 hour to denature the protein.

  The resulting solution was loaded onto a column containing nickel nitrotriacetic agarose (Qiagen, Hilden, Germany) to bind the protein. After washing with buffer C (the above-mentioned buffer B containing 1% Tween but not containing β-ME), the WT1 partial protein was buffered with Buffer D (containing 150 mM imidazole but not containing β-ME, pH 8.0, pH 8.0). Elution with 4 ml). To refold the recombinant protein, the eluate is placed in a cassette (Slide-a-Lyzer cassette, Pierce Chemical Company, IL) and overlaid with an excess of 20 mM Tris-HCl (pH 8.0) buffer at 4 ° C overnight. Dialyzed against.

The recombinant protein was then concentrated using Centricon-30 (Milipore Corp., Bedford, Mass.), And the protein concentration was measured by the Bloodhod method using a protein analysis kit (Bio-Rad Labs., Hercules, Calif.). The purity and specificity of the obtained protein were confirmed by SDS-PAGE and Western blot analysis, and stored as a 30% (v / v) glycerol solution at −80 ° C. until use.
(3) Measurement of WT1 antibody The WT1 partial protein (HWT3) obtained above was transformed into a nitrocellulose membrane (Optitran, Shleicher
& Schuell) at a concentration of 2.5 μg / cm ² (1 hour incubation at room temperature). The membrane was washed with PBS, blocked in PBS containing 1% BSA for 2 hours, and mounted on a dot blot apparatus (Schleicher & Schuell, Dassel, Germany) according to the specifications.

  The measurement was performed according to Example 2. That is, 20 μl of the test serum prepared in (1) above (diluted 1/500 in the case of IgM in PBS containing 1% BSA and 0.1% Tween 20 and 1/2500 in the case of IgG) was added to each well. And incubated at room temperature for 1 hour. After washing with PBS, the membrane was further reacted with HRP-conjugated goat anti-human IgM antibody or HRP-conjugated rabbit anti-human IgG antibody for 1 hour at room temperature in 1% BSA / PBS.

After sufficiently washing with PBS, the antibody titer was determined in the same manner. The result was obtained as an average value of at least two tests.
(4) Results
(a) The results obtained according to the above method (measurement results of IgM or IgG type WT1 antibody of the specimen) are shown in Table 1 and FIG. 8 (measurement results of IgM type WT1 antibody) and FIG. 9 (measurement results of IgG type WT1 antibody). ).

In each figure, the vertical axis indicates antibody titer (Densitometric Units), and the horizontal axis indicates each specimen. In each figure, black circles indicate specimens having IgM or IgG type WT1 antibodies alone, and white circles indicate specimens having both IgM type WT1 antibodies and IgG type WT1 antibodies. The cutoff value of the WT1 antibody was an antibody titer of 600 for an IgM type antibody and an antibody titer of 500 for an IgG type antibody (Clin. Chem., 39 , 561 (1993)).

  From the results shown in Table 1 and FIGS. 8 and 9, the following can be understood.

  That is, IgM type WT1 antibody was detected in 24 (52.2%) of 46 patients with WT1-related diseases. On the other hand, it was detected in 7 (16.2%) of 43 healthy subjects.

  Both the IgM type WT1 antibody detection rate (p = 0.0001) and the IgM type WT1 antibody titer (p <0.0001) were significantly higher in WT1-related disease patients than in healthy subjects.

  As a result of comparing IgM-type WT1 antibody titers in four types of WT1-related diseases with healthy subjects, it was found that the antibody titers in patients with WT1-related diseases except ALL were significantly higher than those in healthy subjects.

  IgG-type WT1 antibody was detected in 23 (50.0%) of 46 patients with WT1-related diseases, but only 2 (4.7%) of 43 cases were detected in healthy subjects.

  Both IgG type WT1 antibody detection rate (p = 0.0001) and IgG type WT1 antibody titer (p <0.0001) were significantly higher in WT1-related disease patients than in healthy subjects. As a result of comparing IgG-type WT1 antibody titers in WT1-related diseases with healthy individuals, it was found that the antibody titers were significantly higher in three types except ALL, namely AML, CML, and MDS, compared with healthy subjects. It was.

  The production of both IgM-type and IgG-type WT1 antibodies shows a marked contrast between WT1-related disease patients and healthy individuals. Among 46 patients with WT1-related disease, 10 (21.7%) produced both IgM and IgG WT1 antibodies, whereas similar cases were not found in 43 healthy subjects. It should be noted that 3 cases (2 AML and 1 MDS) that had the highest antibody titer of IgG type WT1 antibody simultaneously produced IgM type WT1 antibody, and in 16 AML patients 6 cases (37.5%) and 4 out of 16 MDS patients (25.0%) produced IgM and IgG type WT1 antibodies simultaneously, whereas both ALL and CML patients received both antibodies. Nothing was produced at the same time.

No correlation was observed between the WT1 antibody titer and the WT1 expression level (RT-PCR) or patient age, and between the presence of the WT1 antibody and the patient's sex, patient pathology and life / death.
(b) WT1 antibody titers were measured in 4 leukemia patients both at diagnosis and at the time of continued complete remission (CCR).

  The results are shown in Table 2.

From the results shown in Table 2, in all these patients, the relatively high WT1 antibody titer detected at the time of diagnosis was not detected at the time of CCR. These patients have maintained CCR for 3.1-5.5 years, have normal levels of serum IgM, IgG, and IgA and have not received any immunosuppressive drugs at the time of the study. This finding indicates that these patients have fully recovered from the immunosuppressed state due to powerful chemotherapy or allogeneic bone marrow transplantation. Therefore, the disappearance of the WT1 antibody in CCR is considered to be due to the disappearance or reduction of the leukemic tumor burden and the subsequent disappearance of immune stimulation by the WT1 antigen.
(c) WT1 antibody class switch
IgM type and IgG type WT1 antibodies were measured in 16 MDS patients (6RA, 4RAEB and 6RAEB-t). The results are shown in FIG.

  The following can be seen from the results shown in the figure. That is, IgM type WT1 antibody was detected in 5 out of 6 RA patients, and 3 out of the 5 cases had relatively high antibody titers. In contrast, IgG type WT1 antibody was not detected in 4 out of 6 cases, and the antibody titers in the remaining 2 cases were also low.

  In RAEB patients, IgM type WT1 antibody and IgG type WT1 antibody were detected in 2 and 3 cases, respectively. The IgM type WT1 antibody titer was lower than those of the RA patients, and the IgG type WT1 antibody titer was high.

  RAEB-t patients were clearly different from RA patients. Two of the 6 cases had low titer IgM WT1 antibodies, while 5 of 6 cases produced high titer IgG WT1 antibodies.

  These facts strongly suggest that an immunoglobulin class switch of the WT1 antibody from IgM to IgG occurs in association with the progression from RA to RAEB to RAEB-t in the pathology of MDS.

Specificity confirmation test of WT1 antibody detection system
(5-1) Place the test protein (HWT3, albumin (HSA) or human transferrin) in each of IgG type WT1 antibody positive specimen serum, IgG type WT1 antibody negative specimen serum and WT1 polyclonal antibody solution (S-Cruz180). Quantification was added and WT1 antibody measurement (Inhibition Assay) was performed according to the method described in Example 4.

  The results are shown in FIG.

  In FIG. 11, the vertical axis shows% inhibition (% inhibition), the horizontal axis shows the amount of test protein added (ng / well), (1) shows WT1 antibody positive specimen + HWT3, (2) shows WT1 antibody Positive sample + HSA, (3) WT1 antibody positive sample + transferrin, (4) WT1 antibody negative sample + HWT3, (5) WT1 antibody negative sample + HSA, (6) WT1 antibody negative sample + transferrin (7) shows WT1 polyclonal antibody solution + HWT3, (8) shows WT1 polyclonal antibody solution + HSA, and (9) shows WT1 polyclonal antibody solution + transferrin.

  From the results shown in FIG. 11, when the HWT3 protein, which is the antigen of the detection system, was added to the WT1 antibody-positive specimen and the WT1 polyclonal antibody solution, the detection of the WT1 antibody was suppressed depending on the amount of HWT3 protein added. This addition-dependent suppression by HWT3 could be confirmed in the same manner when an IgM type WT1 antibody-positive specimen was used. On the other hand, the addition of HSA and transferrin did not show such suppression, and the WT1 antibody-negative sample was not affected by the addition of HWT3.

From these results, it was confirmed that the present WT1 antibody detection system specifically detects an antibody against WT1.
(5-2) K562 (erythroleukemia cell line) nuclear lysate was subjected to 10% SDS-PAGE to prepare WT1 protein (natural WT1 protein) for Western blot analysis. Using the IgG-type WT1 antibody positive sample and the same negative sample measured in Example 4, Western blot analysis of the WT1 protein was performed. The sample serum was diluted 50 times, and ALP-labeled anti- (rabbit or human) IgG antibody was used as the second antibody. Moreover, WT1 polyclonal antibody solution (S-Cruz180) was used as a positive control.

  The results are shown in FIG.

  In FIG. 12, the position of WT1 is shown in the left column together with the size marker.

  Lane 1 shows the results using the positive control (S-Cruz180), Lane 2 shows the results using the IgG-type WT1 antibody positive (ALL) sample measured as the antibody titer 1366 in Example 4, and Lane 3 shows the same antibody. Results using an IgG type WT1 antibody positive (AML) sample measured as 754, Lane 4 is a result using an IgG type WT1 antibody negative (RAEB-t) sample that was below the cutoff value in Example 4 Similarly, lanes 5 and 6 show the results of using a healthy subject negative for IgG type WT1 antibody, respectively.

  From the results shown in FIG. 12, it can be seen that natural WT1 protein can be detected with sera positive for WT1 antibody in the detection system of the present invention (it cannot be detected with the same negative serum). It was confirmed that a desired WT1 antibody that recognizes the WT1 protein was detected.

1 is a diagram schematically showing the structure of a WT1 antigen produced according to Example 1 (1). FIG. 2 is a drawing-substituting photograph showing the results of a reactivity test between a WT1 antigen and a WT1 antibody according to Example 1, (2). 3 is a graph showing WT1-related diseases and healthy subject WT1 antibody titers (IgG) determined by the test method of the present invention according to Example 2. FIG. 3 is a graph showing WT1-related diseases and healthy subject WT1 antibody titers (IgG) determined by the test method of the present invention according to Example 2. FIG. 3 is a graph showing WT1-related diseases and healthy subject WT1 antibody titers (IgM) determined by the test method of the present invention according to Example 2. FIG. 3 is a graph showing WT1-related diseases and healthy subject WT1 antibody titers (IgM) determined by the test method of the present invention according to Example 2. FIG. 6 is a graph showing changes in WT1 antibody titers before the start of treatment for acute leukemia and by complete remission obtained by the test method of the present invention according to Example 3. 6 is a graph showing WT1-related diseases and healthy subject WT1 antibody titers (IgM) determined by the test method of the present invention according to Example 4. 6 is a graph showing WT1-related diseases and healthy subject WT1 antibody titers (IgG) determined by the test method of the present invention according to Example 4. 6 is a graph showing WT1 antibody titers (IgG and IgM) of WT1-related diseases determined by the test method of the present invention according to Example 4. FIG. 6 is a graph showing the specificity of a WT1 antibody detection system tested according to Example 5 (1) and according to the test method of the present invention. 6 is a drawing-substituting photograph showing the results of Western blot analysis tested according to Example 2 (2).

Claims (8)

A test method for determining the presence or treatment progress and prognosis of a WT1-related disease in a subject, characterized by measuring the amount of antibody against WT1 in a specimen and using the measured value as a clinical index in the test Test method for WT1-related diseases. The test method according to claim 1, wherein the measurement of the amount of antibody against WT1 is performed by an immune reaction using the WT1 antigen. The test method according to claim 2, wherein the WT1 antigen is a WT1 protein that contains a suppressor region and an activation region and lacks a zinc finger. 2. The test method according to claim 1, wherein the antibody against WT1 is an IgG antibody or an IgM antibody. 2. The test method according to claim 1, wherein a change with time in the amount of antibody against WT1 is a clinical index. 2. The test method according to claim 1, wherein the ratio of IgG antibody to IgM antibody against WT1 is a clinical index. 2. The examination method according to claim 1, wherein the progression from myelodysplastic syndrome to leukemia is determined. 2. The test method according to claim 1, wherein a complete remission of leukemia is determined.

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