JPH0843384A - Diagnosing method for large intestine cancer and diagnostic medicine - Google Patents

Abstract

PURPOSE:To sense a patient suffering from a large intestin decease at an early stage with a high rate of positiveness by measuring the existing amount of a blood vessel penetrative factor (VPF) in a serum using an antibody to the VPF, and diagnosing existence of large intestin cancer on the basis of the amount obtained. CONSTITUTION:By a conventional method, a monochronal antibody and a polychronal antibody for human VPF 121 are acquired, and with them the VPF in the serum is measured by the enzyme immune measuring method. A significant difference exists in the VPF SENSING amount between a healthy person and a patient with large intestin patient, and if analysis is made stage by stage, there is a patient who has a high VPF regardless of stage. and therefore, it is judged that diagnosis can be passed at all stages. If the rate of positiveness in diagnosis is determined while the value twice of the sum of the meaz of the measurements from a healthy person and the standard deviation is used as a cutoff value, a much higher rate of positiveness is exhibited than the value according to the conventional diagnosis for large intestin cancer, so that this should favorably be used as a diagnostic medicine for large intestin cancer.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and a method for diagnosing colorectal cancer characterized by measuring the abundance of a blood vessel permeability factor (hereinafter referred to as VPF) in serum and making a diagnosis based on the measured value. The present invention relates to a diagnostic agent for colorectal cancer, which comprises a VPF antibody used in. V in serum
The colorectal cancer diagnosis of the present invention by measuring the abundance of PF can detect colorectal cancer patients at a much higher rate than in the case of using various known tumor markers and occult blood test of stool, which is clinically very important. Therefore, the present invention is useful in the field of medicine, particularly in the field of diagnostic agents, and is widely used in those fields.

[0002]

2. Description of the Related Art Currently, the number of patients with colorectal cancer in Japan is increasing year by year, and a diagnostic method for early detection is required. Colorectal cancer progresses relatively slowly, and even with advanced cancer, if the curative resection is completely performed, the prognosis is relatively good.
There are few subjective symptoms until it progresses to a considerable extent, and it is often the case that metastases and infiltration have already occurred at the time of definitive diagnosis, making it impossible to excise. The fecal immunooccult blood reaction and various tumor markers are used for the diagnosis of colorectal cancer, but the positive rate is not satisfactory in any of them. That is, the positive rate of the immunooccult blood test of stool used for the diagnosis of colorectal cancer is 50.
~ 60%, Carci as a tumor marker for colorectal cancer
noembryonic antigen (CEA), CA19-9, NCC-
There are ST-439, STN, etc., which are used to judge the therapeutic effect and monitor recurrence, but the tumor marker positive rate by stage is CEA even in Dukes C where curative resection is possible.
36 for CA19-9, NCC-ST-439, STN
%, 30%, 35%, and 21%, which are not sufficient tumor markers for the detection of early colorectal cancer (Ohisa Hisao et al. Tumor marker for CRC. CRC
1 (4), 42-47 (1992)).

[0003]

DISCLOSURE OF THE INVENTION The present inventors have conducted earnest studies to find a method for early diagnosis of colorectal cancer that can detect colorectal cancer patients at a very high rate as compared with conventional methods. Of.

[0004]

Means for Solving the Problems As a result of various investigations, the present inventors have found that VPF in serum between a colon cancer patient and a healthy subject.
It was found that there is a significant difference in the abundance of
It was found that colorectal cancer can be diagnosed with a positive rate of 80% or more even in the early stage of colorectal cancer if the colorectal cancer is diagnosed based on the amount of PF present, and the present invention was completed. That is, the present invention measures the abundance of VPF in serum,
An invention relating to a method for diagnosing colorectal cancer characterized by diagnosing based on the measured value, and further measuring the abundance of VPF in serum using an antibody against VPF, and diagnosing based on the amount The present invention relates to a method for diagnosing colorectal cancer and a diagnostic agent for colorectal cancer characterized by comprising an antibody against a vascular permeability factor.

The present invention will be described in detail below. The present invention is to measure the abundance of VPF in serum using an antibody against VPF, particularly an antibody against human VPF, and to diagnose colorectal cancer based on the amount.
VPF, or vascular permeability f
actor is vascular endothelium
It is the same factor as that called ial cell growth factor (VEGF), and is one of the factors known to induce angiogenesis, like the following factors. That is, in addition to VPF, other known factors that induce angiogenesis include basic fibroblast growth factor (basic fibroblas growth factor) as a substance that directly acts on vascular endothelial cells.
t growth factor, bFGF), acidic fibroblast growth factor
(acidic fibroblast growth factor, aFGF), platelet-derived endothelia
cell growth factor (PD-ECGF), etc., and transf as a substance indirectly acting on vascular endothelial cells.
orming growth factor-α (TGF-α), transforming g
rowth factor-β (TGF-β), angiogenin, tumor necr
osis factor-α (TNF-α) etc. [Folkman, J. & Shi
ng, Y., J. Biol. Chem., 267: 10931 (1992)]. These factors play an important role in physiological or pathological phenomena such as fetal growth, wound healing, and cancer cell proliferation by inducing angiogenesis, that is, proliferation, migration and invasion of capillary endothelial cells into tissues. Known to play
[(Folkman, J., Cancer Res. 46: 467 (1986)].

Of these factors that induce angiogenesis, V
Regarding PF, it is secreted in normal or tumor cell lines of mouse, rat, guinea pig, bovine and human, and has been shown to be present in the brain, pituitary gland, kidney and ovary by histology [(Ferrara , N., Et.al. Endocrine R
eviews 13:18 (1992)]. Furthermore, regarding human VPF,
Angiogenesis and metastasis of breast cancer [Weider, N, et.al. N. Engl. J. Me
d. 324: 1 (1991)] and angiogenesis of renal cell carcinoma [History of Medicine, 1
68: 231 (1994)], or angiogenesis in retinal disease [Ad
amis, AP et.al., Biochem.Biophys.Res.Comm., 193: 63
1 (1993)]. Also,
VPF is a receptor (flt, fms-lik) existing on the surface of target cells.
It has been clarified that a signal is transduced into the cell by binding to eTyrosine kinase) [De Vies,
C. et. Al. Science, 255: 989 (1992)], the mechanism of interaction between VPF and its receptor and the signal transduction mechanism have not been elucidated in detail.

CDNA of the human VPF gene
Has been isolated, the base sequence has been determined, and the amino acid sequence has been deduced. This gene consists of four types of proteins with different number of amino acid residues (1 amino acid residue is
21 kinds, 165 kinds, 189 kinds, and 206 kinds) were produced, and among them, those having 121 amino acid residues (hereinafter also referred to as VPF ₁₂₁ and other things) and VP
F ₁₆₅ is said to be a mature protein [(Ferrara, N.,
et.al. Endocrine Reviews 13:18 (1992)]. VPF ₁₂₁
Although one in which 44 amino acids at the carboxyl terminus of VPF ₁₆₅ is deficient, while the VPF ₁₂₁ and VPF _165, is not clear as to whether there is a difference in the effect on vascular endothelial cells.

[0008] The measurement of the amount of VPF in serum is known as V
It can be carried out by an enzyme immunoassay using a monoclonal antibody against PF, etc., and the antibody used therefor can also be prepared by a known method, and there are many reports that an antibody for VPF was obtained (K. Jin Kim and others NATURE VOL3
62 29 APRIL 1993 841-844). The present inventors also used human VP
We obtained several kinds of monoclonal antibodies against F ₁₂₁ , and also obtained those monoclonal antibodies and human VPF _121.
The present inventors have clarified that VPF can be measured by an enzyme immunoassay using a polyclonal antibody against Escherichia coli (patent application dated June 10, 1994; title of invention: "peptide and monoclonal antibody", etc.).

[0009]

As described above, it is known that factors involved in angiogenesis play an important role in the growth of cancer cells. Furthermore, regarding human VPF, angiogenesis and metastasis of breast cancer and renal cell carcinoma Although known to be involved in angiogenesis, VPF is secreted in both normal and tumor cell lines of mouse, rat, guinea pig, bovine and human, and cancer patients, especially large intestine. It is unclear why there is a specific difference in the abundance of VPF in serum between a cancer patient and a healthy person. However, based on the difference found by the present invention, it is possible to diagnose cancer, especially for early cancer. Diagnosis can be performed at a higher rate than before.

[0010]

Embodiments will be described in more detail based on the following embodiments. Example 1 (1) Preparation of VPF Monoclonal Antibody and VPF Polyclonal Antibody VPF monoclonal antibody and VPF polyclonal antibody were prepared by using isolated human VPF cDNA as a fusion protein (GST) with glutathione S-transferase (GST).
-VPF) was produced in E. coli, and the obtained protein was used as an antigen to prepare a mouse monoclonal antibody according to a conventional method. That is, GST-VPF (100 μg) was mixed with Freund's complete adjuvant in an equal amount, and intraperitoneally administered to BALB / C mice on day 0, 14 days, and 25 days after administration of 3 doses, immunized mouse splenocytes and mouse myeloma cells. (SP2) was fused in the presence of PEG by incubating for 1 minute. The obtained cells are HA
T medium [composition: hypoxanthine (1 × 10 ⁻⁴ M), aminopterin (4 × 10 ⁻⁷ M), thymidine (1.6 × 10 4)
^-5 M), penicillin (100 units / ml), fetal bovine serum (20
%), Streptomycin (100 μg / ml), 2-mercaptoethanol (2 × 10 ⁻⁵ M) in RPMI1640
Hybridomas were selected by culturing in a medium. The obtained hybridoma was cloned by the limiting dilution method. On the other hand, yeast which produces VPF was isolated from V
A circular DNA containing PF cDNA was prepared by introducing it into yeast Saccharomyces cerevisiae by the lithium acetate method, and cation exchange chromatography (TSK-SP650 manufactured by Tosoh Corporation), ammonium sulfate precipitation and gel filtration chromatography were performed from the culture solution of this yeast. Yeast-derived human VPF (hereinafter referred to as YVPF) was prepared by using a graph (Superdex-75 manufactured by Pharmacia) (patent application;
-200181). The yeast producing VPF was deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, under the deposit numbers FERM P-13730 and FE.
RM P-13731 is provided. This YVPF
The reactivity of the culture supernatant of the cloned hybridoma was examined by an enzyme immunoassay, and a hybridoma producing a monoclonal antibody that reacts with YVPF was selected. The obtained hybridoma producing the monoclonal antibody was deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, under the deposit numbers FERM P-14345 and FERM.
P-14346 and FERM P-14347 are provided.

(2) Preparation of VPF Monoclonal Antibody The selected hybridoma was intraperitoneally transplanted into a nude mouse, and ascites containing a large amount of the monoclonal antibody was collected.
The obtained ascites fluid was used for protein G affinity column (MAb
A monoclonal antibody purified by treatment with Trap GII (Pharmacia) was obtained, and one of the monoclonal antibodies was named MV303 and used in the following experiments. As a result of examining the class of the MV303 antibody by an enzyme immunoassay using an anti-mouse immunoglobulin subclass-specific antibody, it was IgG2a. This monoclonal antibody MV303 recognized a peptide having an amino acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2 showing a part of the amino acid sequence of VPF.

(3) Preparation of Anti-VPF Polyclonal Antibody GST-VPF was used as an antigen to immunize rabbits by a conventional method. Rabbit serum with increased antibody titer was separated and treated by anion exchange column chromatography to obtain an IgG fraction of rabbit anti-VPF polyclonal antibody.
A part of the IgG fraction was digested with pepsin to prepare F (ab ') _2, which was then bound to peroxidase by the maleimide method.
A rabbit anti-VPF polyclonal antibody labeled with peroxidase was obtained.

(4) Measurement of VPF content in serum of colorectal cancer patient by enzyme immunoassay 1) Measurement by colorimetric method Construction of method for measuring VPF content in serum using rabbit anti-VPF polyclonal antibody and MV303 antibody did.
That is, 96 μl of 5 μg / ml anti-VPF polyclonal antibody
Place 100 μl of each in the plate for enzyme immunoassay at 4 ℃
The plate was left to stand overnight in the plate to adsorb the anti-VPF polyclonal antibody to the plate. Phosphate buffered saline containing 0.1% bovine serum albumin (hereinafter, bovine serum albumin is BSA, phosphate buffered saline is PBS, and phosphate buffered saline containing bovine serum albumin is B
It is called SA / PBS, and the concentration of BSA is shown at the beginning of the word.
After washing the wells of the plate 6 times with 1% BSA / PBS), 1% BSA / PBS was placed in the wells and left at room temperature for 1 hour. After removing 1% BSA / PBS from the hole, P
Serum diluted 2-fold with BS was added and left at room temperature for 1 hour. After washing 6 times with 0.1% BSA / PBS, 5 μg / ml M
100 μl of V303 antibody (0.1% BSA / PBS solution)
Each of these was placed and left at room temperature for 1 hour. 0.1% BSA / PB
After washing 6 times with S solution, peroxidase-labeled goat anti-mouse immunoglobulin (0.1% BSA / PBS solution) was added and left at room temperature for 1 hour. After washing 6 times with 0.1% BSA / PBS, 0.15 M citrate buffer containing 0.2 mg / ml orthophenylenediamine and 0.015% hydrogen peroxide.
(pH = 5.0) was added for color development. The reaction was stopped by adding 10% sulfuric acid, and then the absorbance (OD490 / 650) was measured.
Similarly, the amount of VPF in the serum of healthy subjects was also measured and compared with the results of colorectal cancer patients. When the above results were plotted on a graph and shown in FIG. 1, a clear significant difference was observed between a healthy subject and a colon cancer patient. Further, the measurement results were analyzed for each stage and shown in FIG. 2. As is clear from the figure, there were patients with high VPF amount regardless of stage. From this, it was clarified that colorectal cancer patients at all stages can be diagnosed by measuring the amount of VPF in serum. The positive rate for diagnosis was determined using the cut-off value as a value that is twice the mean value + the standard deviation of the measured values for healthy subjects, and is shown in Tables 1 and 2. As is clear from those tables, the positive rate by the method of the present invention is the positive rate (50% to 60%) of the existing colon cancer diagnosis.
%), Which is much higher than 50% to 100%, indicating that the colorectal cancer diagnosis method of the present invention is very useful.

[0014]

[Table 1]

[0015]

[Table 2]

2) Measurement by luminescence method 100 μl / we of VPF polyclonal antibody (5 μg / ml)
ll each in a 96-well plate and leave at 4 ° C overnight,
It was washed 4 times with 0.1% BSA / PBS. 1% BSA /
After blocking with PBS (1 hour at room temperature), sera of colon cancer patients and healthy subjects (diluted 2-fold with PBS) were added and reacted at room temperature for 1 hour. 0.1% BSA / PBS
After washing 4 times with MV303 antibody (5 μg / ml of 0.1% BSA
/ PBS solution) was added at 100 μl / well and reacted at room temperature for 1 hour. After washing 4 times with 0.1% BSA / PBS, alkaline phosphatase-labeled anti-mouse IgG (0.1% B
Diluted 1000 times with SA / PBS) 100 μl /
Wells were put in each well and reacted at room temperature for 1 hour. 0.1% BSA
After washing 4 times with PBS / PBS, 0.115 mg / ml AMPPD (4-
Methoxy-4- (3-phosphatephenyl) spiro [1,2-dioxetane-
3,2'-adamantane], disodium salt) 50 mM carbonate buffer
(pH = 9.5) was added at 100 μl / well and left at room temperature for 20 minutes, and the luminescence intensity was measured. The results measured by the above method are plotted in a graph and shown in FIG. 3, and the measurement results are analyzed for each stage and shown in FIG. 4, as in the case of the colorimetric method. As is clear from the above, patients with high VPF amount were observed regardless of stage. From this, it was revealed that colorectal cancer patients at all stages can be diagnosed by measuring the amount of VPF in serum by a luminescence method. As in the case of the colorimetric method, the positive rate of diagnosis was determined and shown in Tables 1 and 2 with the cut-off value being the value of the mean value + the standard deviation of the measured values of healthy subjects. As is clear from these tables, the positive rate by the method of the present invention is much higher than the positive rate (50% -60%) in the existing colorectal cancer diagnosis.
% To 100% regardless of stage, showing that the method for diagnosing colorectal cancer of the present invention is very useful. From the above, it was clarified that colorectal cancer patients at all stages can be diagnosed by measuring the amount of VPF in serum, and that the luminescence method can diagnose colon cancer at a higher rate than the colorimetric method.

Comparative Example 1 In the same manner as in Example 1, VPF in serum of cervical cancer patients
The amount was measured, and as shown in Table 3, the luminescence method showed a positive rate of about the level of a conventional tumor marker, but the colorimetric method showed a low positive rate, which was not superior to that of the conventional product. Two of the healthy subjects showed an abnormally high VPF amount, but considering the numerical value, it is presumed that there is some other cause, so they were excluded from the samples.

[0018]

[Table 3]

[0019]

[Effect of the Invention] Regardless stage by measuring the serum VPF amount using the enzyme immunoassay using a polyclonal antibody against the monoclonal antibody and a human VPF ₁₂₁ against human VPF _121, conventional tumor markers and stool occult blood The present invention, which has an excellent effect that it is possible to diagnose colorectal cancer patients at a much higher rate than when using a reaction, and which is a diagnosis based on the amount of VPF in serum, is used for early detection of colorectal cancer and pathological conditions of colorectal cancer. It is excellent because it can be widely used for judging the progress and treatment effect, and predicting recurrence.

[0020]

[Sequence list]

SEQ ID NO: 1 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin: Cell line: SEQ ID NO: 2 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin: Cell line:

[Brief description of drawings]

FIG. 1 is a diagram showing the results of measuring the amount of VPF in serum of colorectal cancer patients and healthy individuals by an enzyme immunoassay method (color development method).

FIG. 2 is a diagram in which the results of FIG. 1 are analyzed for each stage of colorectal cancer patients.

FIG. 3 is a diagram showing the results of measuring the amount of VPF in serum of colorectal cancer patients and healthy individuals by an enzyme immunoassay method (luminescent method).

FIG. 4 is a diagram in which the results of FIG. 3 are analyzed for each stage of colorectal cancer patients.

Claims (3)

[Claims] 1. A method for diagnosing colorectal cancer, which comprises measuring the abundance of vascular permeability factor in serum and diagnosing based on the measured value. 2. The method for diagnosing colorectal cancer according to claim 1, wherein the abundance of the vascular permeability factor is measured using an antibody against the vascular permeability factor. 3. A diagnostic agent for colorectal cancer, which comprises an antibody against a vascular permeability factor.