JP7106810B2 - Novel lung cancer marker - Google Patents

Description

The present invention relates to diagnostic technology, and more particularly to lung cancer diagnostic technology.

Tumor markers in biological samples, particularly in serum, are used as auxiliary cancer diagnostic methods such as cancer screening, prognostic criteria after surgery, and therapeutic efficacy criteria. As lung cancer markers, CYFRA21-1, CEA, SCC, SLX (Sialyl Lewis X), CA125, etc. have been used, but they cannot be said to have high sensitivity and specificity, and lung cancer has better sensitivity and specificity. A marker was desired.

Non-Patent Document 1 reports that gene expression of CHL1 (close homolog of L1), which is a cell adhesion molecule, was investigated in various cancers including lung cancer.
However, the gene expression analysis in the literature shows that CHL1 is both increased and decreased in lung cancer, and the relationship between CHL1 and lung cancer is unclear. In addition, from analysis at the gene level, it was unclear whether CHL1 protein was expressed in lung cancer tissues, and the leakage of CHL1 protein from lung cancer tissues into serum was also unknown.

PLoS ONE, 6, 3, e15612 (2011)

An object of the present invention is to provide a novel marker for lung cancer diagnosis.

The present inventors have developed a mouse lung cancer model EML4 (echinoderm microtubule-associated protein-like 4)-ALK (a
As a result of analysis using napplastic lymphoma kinase)-TG (transgenic) mice, CHL1 was present not only in exosomes but also in serum as its truncated form, and more CHL1 was detected than in wild-type mouse serum. I found out.

In addition, as a result of comparing the serum CHL1 level in mouse lung cancer model EML4-ALK-TG mice with wild-type mice, it was found that TG mice were significantly elevated. was found to increase in proportion to .

Furthermore, when the serum of human lung cancer patients was also examined, it was found that the serum CHL1 level in lung cancer patients was higher than the average serum CHL1 level in healthy subjects.

Based on the findings as described above, the inventors completed an invention relating to a data acquisition method for diagnosing lung cancer by measuring the amount of CHL1 protein in a biological sample.

That is, the present invention is as follows.
[1] A data acquisition method for diagnosing lung cancer, comprising the step of measuring the amount of CHL1 protein in a biological sample derived from a subject.
[2] The method of [1], wherein the method is for diagnosing lung cancer based on the criteria that if the amount of CHL1 protein is higher than the level in a healthy subject, the possibility of having lung cancer is high.
[3] The method of [1] or [2], wherein the lung cancer is non-small cell lung cancer.
[4] The method according to any one of [1] to [3], wherein the lung cancer is EML4-ALK mutant lung cancer.
[5] The method according to any one of [1] to [4], wherein the biological sample is serum, plasma, or an extracellular vesicle fraction thereof.
[6] The method according to any one of [1] to [5], wherein the step of measuring is by immunoassay. [7] The method according to [6], wherein the step of measuring is by an ELISA (enzyme-linked immunosorbent assay) method.
[8] The method according to any one of [1] to [7], wherein the subject is a mouse or a human.
[9] A diagnostic kit for lung cancer, comprising a reagent for measuring the amount of CHL1 protein.
[10] The kit of [9], wherein the reagent for measuring the amount of CHL1 protein contains at least an anti-CHL1 antibody.

ADVANTAGE OF THE INVENTION According to this invention, the marker for highly accurate lung cancer diagnosis is provided. When measuring CHL1 in serum, a minimally invasive test is possible.

In addition, lung cancer caused by the EML4-ALK fusion gene (EML4-ALK mutant lung cancer) requires a test method that takes time for its diagnosis and assessment of therapeutic effects, so the CHL1 molecule is a new tumor marker for this EML4-ALK mutant lung cancer. If so, it is possible to significantly reduce the inspection time.

Fig. 2 is a diagram confirming the presence of CHL1 protein in serum samples of EML4-ALK-TG mice by Western blotting (photograph). Detection was performed in the extracellular vesicle fraction (upper panel) and the serum supernatant (lower panel) of wild-type mice (WT) and EML4-ALK-TG mice (TG). The portion indicated by the arrow is mouse CHL1. Quantification of CHL1 protein in serum of EML4-ALK-TG mice by ELISA method. A comparison of serum mouse CHL1 levels was performed between wild-type mice (WT) and EML4-ALK-TG mice (TG). TG mice were detectable at significantly higher levels than WT mice (p=1.801×10 ⁻⁹ ). FIG. 4 shows the correlation between the amount of CHL1 protein and tumor weight in EML4-ALK-TG mice. Fig. 3 is a diagram confirming the presence of CHL1 protein in human serum samples by Western blotting (photo). Detection was performed in the serum extracellular vesicle fraction (upper figure) and the serum supernatant (lower figure) from healthy subjects and lung cancer patients. The portion indicated by the arrow is human CHL1.

The present invention provides a data acquisition method for diagnosing lung cancer, comprising the step of measuring the amount of CHL1 protein in a biological sample derived from a subject.

A subject to be tested may be any animal that may cause lung cancer, preferably a mammal, more preferably a mouse or a human.

CHL1 (Close homolog of L1) protein is a single transmembrane cell adhesion molecule (CAM), also called CALL (cell adhesion L1-like), which is a cell adhesion molecule L1, and its function in nerve cells is suggested. ing.

The CHL1 protein may be a protein derived from a subject, and specific examples thereof include proteins containing the amino acid sequences shown below. CHL1 protein also includes protein fragments, analogs, and variants that have lung cancer relevance similar to the protein. Examples of protein fragments include extracellular domain fragments.

The amino acid sequence of CHL1 protein is exemplified by the amino acid sequence of SEQ ID NO: 1 when the subject is a mouse. In addition, when the test subject is human, the amino acid sequence of SEQ ID NO: 2 is exemplified. Proteins having amino acid sequences in which one or several (eg, 1 to 20) amino acids are substituted, deleted, inserted or added to these amino acid sequences may also be used. Moreover, when using a different animal as a test subject, a homologous protein derived from the animal is the subject of measurement.

A healthy subject is generally defined as one who does not have a specific chronic disease. In the present invention, the term may be used as defined above, but may also be used to mean a person who does not have lung cancer but has other diseases. In addition, in the present invention, a healthy person and a normal person are synonymous and are terms that can be replaced with each other.

Regarding the expression level of the CHL1 protein, the “level” may be the actual measured value by the above-described measurement method, that is, the measured value, the corrected value corrected by the negative control measured value, or the index value corrected by the relative index. It's okay. These are merely examples, and are not limited to these values, and other values can be adopted.

Lung cancer is classified into non-small cell lung cancer and small cell lung cancer according to histological type. Non-small cell carcinoma is further classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Lung cancer in the present invention may be any of the above classifications. Furthermore, the term "lung cancer" is not particularly limited in terms of disease site, disease stage, etc. in the lung, and includes any disease site, disease stage, and the like.

EML4-ALK mutant lung cancer is a type of non-small cell lung cancer, and in the case of humans, a slight inversion occurs within the short arm of chromosome 2, and as a result, the intracellular region of the receptor tyrosine kinase ALK binds to microtubules. It is a lung cancer characterized by the generation of a new active fusion kinase EML4-ALK fused with the protein EML4 (reference: Journal of the Japanese Internal Medicine Society, Vol. 99, No. 4, pp. 155-159). Here, EML4-ALK mutant lung cancer is a type of non-small cell lung cancer and is found in about 4-5% of non-small cell lung cancer patients.

Biological samples include body fluids, cells, tissues, and the like collected from subjects. Body fluids include blood, lymph, interstitial fluid, body cavity fluid, cerebrospinal fluid, and the like. Among these, blood samples are preferred, and blood includes serum, plasma and the like. Serum and plasma may be used as they are, or separated into a precipitate fraction (extracellular vesicle-containing fraction) and a supernatant fraction, and either of them may be used.

A method for measuring CHL1 protein is not particularly limited, and a known protein measuring method can be used, but an immunoassay method using an antibody against CHL1 protein can be preferably used.
The immunoassay method is preferably a method of quantitative detection according to an enzyme immunoassay method, a method of measurement by a fluorescence immunoassay method, a chemiluminescence immunoassay method, or the like. Enzyme immunoassay is a detection method that uses an enzyme as a labeling substance among labeling immunoassay methods. It is also particularly preferred to choose an ELISA method using an immunosorbent.

Examples of the ELISA method include a direct method, an indirect method, and a sandwich method, and any method can be used.

A direct method in the ELISA method refers to a method well known in the art. That is, it is a method of detecting a labeled substance by binding a labeled antibody to CHL1 in a biological sample immobilized on a solid phase.

An indirect method in the ELISA method refers to a technique well known in the art. That is, after binding a primary antibody to CHL1 in a biological sample immobilized on a solid phase to form an immune complex on the solid phase surface, a labeled secondary antibody that recognizes the primary antibody is used. This is a technique for detecting a labeling substance by

A sandwich method in the ELISA method refers to a method well known in the art. Specifically, CHL1 in a biological sample derived from a subject is captured by an immobilized antibody (primary antibody) immobilized on a solid phase, and a labeled secondary antibody is bound to the captured CHL1. 2) is a technique in which an immunocomplex in which the two types of antibodies are bound is formed on a solid-phase surface, and then a labeling substance is detected. The detection sensitivity may be increased by using an unlabeled secondary antibody and then using a labeled tertiary antibody that recognizes the secondary antibody.
In the sandwich method, the order of forming immune complexes is not particularly limited. The biological sample containing CHL1 and the secondary antibody may be added to the immobilized antibody in that order to bind, or the biological sample containing CHL1 and the secondary antibody may be first mixed to form a complex. may be added to and allowed to bind to the immobilized antibody.

In the ELISA method, each antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody in order to stably supply antibodies of stable quality.
In addition, commonly used antibodies such as those derived from mice, rats, rabbits, goats, sheep, and chickens can be used, but not limited thereto, and any antibody that specifically binds to CHL1 can be used. Available.

Antibodies that recognize the extracellular domain of CHL1 are preferred for measuring CHL1 in samples such as serum. Specifically, in mouse CHL1, an antibody that recognizes a part of the extracellular domain represented by amino acids 1 to 1081 of SEQ ID NO: 1, and in human CHL1, amino acids 1 to 1096 of SEQ ID NO: 2 Antibodies that recognize a portion of the extracellular domain of the antibody are preferred.

As the anti-CHL1 antibody, a commercially available one can be used, or one obtained by a conventional monoclonal antibody production method well known to those skilled in the art can be used.

The labeled secondary antibody is not particularly limited as long as it recognizes the primary antibody. For example, when the primary antibody is a rabbit antibody, a labeled anti-rabbit IgG antibody can be used as the secondary antibody, and when the primary antibody is a mouse antibody, a labeled anti-mouse IgG antibody can be used as the secondary antibody.

Labeling substances include enzymes, radioactive isotopes, fluorescent substances, luminescent substances, colloidal gold, and the like.
Among these, enzymes are preferred from the viewpoint of sensitivity and ease of operation, and horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GOD) and the like are more preferred. When HRP is used as a labeling substance, TMB (3,3',5,5'-tetramethylbenzidine) or the like is used, and when AP is used, AMPPD (3-(2'-spiroadamantane)-4-methoxy-4- (3″-phosphoryloxy)phenyl-1,2-dioxetane disodium salt), 9-(4-chlorophenylthiophosphoryloxymethylidene)-10-methylacridan disodium salt, etc. can be used as substrates. can. In addition, as a labeling substance, FITC (fluorescein isothiocy
Anate), rhodamine, and other fluorescent dyes can also be used.

The method of detecting and quantifying the substance to be measured varies depending on the labeling method, and can be performed by a commonly used method well known to those skilled in the art, and is not particularly limited. For example, when HRP, AP, GOD, or the like is used as a labeling substance, by adding a chromogenic substrate or a luminescent substrate, changes in absorbance or luminescence intensity can be measured to quantify the substance to be measured. Moreover, when a fluorescent substance is used as the labeling substance, the substance to be measured can be quantified by measuring the fluorescence intensity. Moreover, when a radioactive isotope is used as a labeling substance, the substance to be measured can be quantified by measuring the radioactivity. Moreover, when colloidal gold is used as the labeling substance, the substance to be measured can be quantified by measuring the absorbance.
For quantification, for example, a calibration curve (standard curve) is prepared in advance with samples of known concentrations, and the measured values are compared with the calibration curve to calculate the CHL1 concentration in the sample.

The solid phase on which the immobilized antibody is immobilized is not particularly limited as long as it is commonly used in ELISA. A magnetic material etc. are mentioned.

The amount of the immobilized antibody to be immobilized on the solid phase is not particularly limited as long as it does not interfere with the antibody-antigen reaction and detection of the labeled substance, and as long as it is not excessively small relative to the amount of CHL1 in the specimen.
After immobilization of the immobilized antibody on the solid phase, it may be appropriately blocked with skimmed milk, albumin, casein or the like in order to prevent non-specific adsorption.

The concentration of the secondary antibody solution is not particularly limited as long as it does not interfere with the antigen-antibody reaction and as long as it is not excessively low relative to the amount of CHL1 in the specimen.
In the present invention, the time for the antigen-antibody reaction, the temperature for performing the method of the present invention, the composition and pH of the sample or reagent diluent and washing solution, etc. are not particularly limited, and are conditions that are generally applied to ELISA methods. OK.

As a "criteria" for evaluating that there is a high possibility of suffering from lung cancer, there is a high possibility of suffering from lung cancer when the amount of CHL1 protein is higher than the level in healthy subjects, or a certain Criteria for evaluating that there is a high possibility of suffering from lung cancer when a measured value or higher is shown.

It may be combined with other lung cancer markers.

Based on the measurement results of the present invention, lung cancer can be diagnosed, and based on the results, treatment strategies such as administration of therapeutic drugs for lung cancer and lung cancer surgery can be formulated.

Another aspect of the present invention provides kits that aid in diagnosing lung cancer, including reagents for measuring the amount of CHL1 protein.

A kit may include means for isolating a biological sample from a test subject. The means may be a method for isolating a blood sample from a test subject and may include a method for separating serum from a blood sample.
A standard may be included.
Additionally, reagents such as labeled secondary antibodies, substrates if the label is an enzyme, blocking agents such as BSA, and the like can be included.
In addition, package inserts describing procedures and diagnostic criteria may be included.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the scope of the present invention is not limited to the following examples.

<Example 1> Collection of biological samples from EML4-ALK-TG mice

First, several-week-old EML4-ALK-TG mice, which are a mouse lung cancer model, were deeply anesthetized with isoflurane, and after cervical dislocation, intracardiac blood sampling was performed. Tumor tissue was then excised and weighed from the mice.

Serum was collected by centrifuging blood samples.
Further, 25 μL of serum was mixed with 6.3 μL of ExoQuick exosome precipitation solution (System Biosciences, California) and incubated at 4° C. for 15 minutes. Centrifugation was performed at 1500 g for 15 minutes to separate the precipitate containing extracellular vesicles from the serum supernatant. The precipitate containing extracellular vesicles was resuspended in 50 µL of PBS (Phosphate buffered saline).

<Example 2> Confirmation of presence of CHL1 protein in blood samples of EML4-ALK-TG mice by Western blotting Western blotting was performed using (Fig. 1). 2 μL each of the serum and the extracellular vesicle fraction was injected onto a 6% SDS-PAGE gel and subjected to electrophoresis. Lanes 1-7 are WT mice and lanes 8-14 are TG mouse samples. It was then transferred to an Immobilon® PVDF membrane (Merck Millipore, Germany). After blocking the protein-transferred PVDF membrane with 5% skim milk, this PVDF membrane was reacted with an HRP-conjugated anti-mouse CHL1 antibody (AF2147; R&D Systems, Minnesota; 1 μg/mL) at room temperature for 1 hour. The PVDF membrane was then subjected to substrate reaction with Immobilon Western Chemiluminescent HRP substrate (Merck Millipore, Germany). The PVDF membrane was exposed and analyzed by a ChemiDoc MP image analyzer (BIO-RAD, Calif.).
As a result of Western blotting, CHL1 could be detected in the serum extracellular vesicle fraction and the serum supernatant excluding this fraction, and its expression level tended to increase in EML4-ALK-TG mice.

<Example 3> Quantitation of CHL1 protein in serum of EML4-ALK-TG mouse by ELISA method (direct method) The mouse serum obtained in Example 1 was diluted 50-fold with PBS, and CORNING, NY; No. 3369) and incubated at 37° C. for 1 hour. After washing with a 0.05% Tween-containing PBS solution, each well was blocked with 5% skim milk at 37° C. for 1 hour. An HRP-conjugated anti-mouse CHL1 antibody was diluted 3000-fold with a 5% skimmed milk solution, added to each well, and incubated at 37° C. for 1 hour. After washing, HRP substrate solution (SureBlue Reserve; SeraCare, MA) was allowed to react for 10-20 minutes at room temperature. After adding 1N HCl solution to each well, the absorbance was measured (450 nm). To correct for measurement errors between ELISA plates, relative values were determined based on the "Relative Index" results.
The mean relative index for wild-type mice was 0.01865 (standard deviation 0.01051) and the mean relative index for EML4-ALK-TG mice was 0.06325 (standard deviation 0.05861). Figure 2). The small p-value (1.801×10 ⁻⁹ ) indicated a significant difference in serum mouse CHL1 levels between wild-type and EML4-ALK-TG mice.

<Example 4> Comparison of correlation between CHL1 protein amount and tumor weight in EML4-ALK-TG mice Analysis by Spearman's rank correlation coefficient showed that serum mouse CHL1 level and tumor weight were as shown in FIG. It was found that there was a significant correlation between Spearman's rank correlation coefficient was 0.692 (p=7.97×10 ⁻⁶ ).

<Example 5> Collection of biological samples from humans Serum was collected by centrifuging blood samples from healthy individuals and lung cancer patients.
Furthermore, 2.5 μL of ExoQuick exosome precipitating solution was mixed with 10 μL of serum and incubated at 4° C. for 30 minutes. Centrifugation at 1500 g for 10 minutes enabled separation of the precipitate containing extracellular vesicles and the serum supernatant. The pellet containing extracellular vesicles was resuspended in 10 μL of PBS.

<Example 6> Confirmation of presence of CHL1 protein in human blood sample by Western blotting Western blotting was performed using the serum extracellular vesicle fraction obtained in Example 5 and the serum supernatant from which the fraction was removed. (Fig. 4). 2 μL each of the serum and the extracellular vesicle fraction was injected onto a 6% SDS-PAGE gel and subjected to electrophoresis. Lanes 1-7 are healthy subjects and lanes 8-14 are lung cancer patient samples. It was then transferred to an Immobilon® PVDF membrane (Merck Millipore, Germany). After blocking the protein-transferred PVDF membrane with 5% skim milk, this PVDF membrane was reacted with an HRP-conjugated anti-human CHL1 antibody at room temperature for 1 hour. The PVDF membrane was then coated with Immobilon Western Chemiluminescent HRP substrate (Merck
Millipore, Germany). The PVDF membrane was exposed and analyzed by a ChemiDoc MP image analyzer (BIO-RAD, Calif.).
As a result of Western blotting, both in the serum extracellular vesicle fraction and in the serum supernatant excluding this fraction, there was a tendency for CHL1 protein to be increased in lung cancer patients.

The present invention can be applied to general lung cancer tumor marker diagnosis, use in definitive diagnosis of human EML4-ALK mutant lung cancer, and use in assays for therapeutic efficacy of human EML4-ALK mutant lung cancer. be done. Applications in the fields of liquid biopsy and precision medicine are also possible.

Claims (9)

A data acquisition method for diagnosing lung cancer, comprising the step of measuring the amount of CHL1 protein in a biological sample derived from a subject , wherein the biological sample is serum, plasma, or an extracellular vesicular fraction thereof . 2. The method according to claim 1, wherein the method is for diagnosing lung cancer by the criterion that if the amount of CHL1 protein is higher than the level in healthy subjects, then the possibility of having lung cancer is high. 3. The method of claim 1 or 2, wherein the lung cancer is non-small cell lung cancer. The method of any one of claims 1-3, wherein the lung cancer is an EML4-ALK mutant lung cancer. The method according to any one of claims 1 to 4 , wherein the measuring step is by immunoassay. 6. The method according to claim 5 , wherein the step of measuring is by an ELISA (enzyme-linked immunosorbent assay) method. The method according to any one of claims 1 to 6 , wherein the subject is a mouse or a human. in a biological sample derived from a test subject containing reagents for measuring the amount of CHL1 proteinwherein the biological sample is serum, plasma, or an extracellular vesicle fraction thereof;Lung cancer diagnostic kit. 9. The kit of claim 8 , wherein the reagent for measuring the amount of CHL1 protein comprises at least an anti-CHL1 antibody.

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