AU2017239516B2 - Method for detection of cancer - Google Patents

Abstract

A method for detecting a cancer(s) based on an expression of prescribed polypeptides is disclosed. These polypeptides were isolated, by the SEREX method using a cDNA library derived from canine testis and serum from a cancer-bearing 5 dog, as a polypeptide which binds to an antibody existing in serum derived from cancer-bearing living body. Because these polypeptides react with antibodies specifically existing in serum of a cancer patient, cancers in a living body can be detected by measuring the antibody in a sample. Cancers in a living body can also be detected by measuring the antigen protein of the antibody per se or mRNA 10 encoding it.

Description

[0001]

The present invention relates to a novel method for detecting a cancer(s).

BACKGROUND ART [0002]

Cancers are the commonest cause for death among all of the causes for death, and the main therapies therefor are palliative treatment in which surgical treatment is 10 combined with radiotherapy and chemotherapy. By virtue of the advance in medical technology, cancers have become such diseases that can be highly possibly cured if they can be found in the early stage. Therefore, a detection method of cancer which can be easily carried out by testing serum, urine or the like without physical and economical burden to patients is demanded.

[0003]

Recently, methods wherein tumor products such as tumor markers are measured have been widely used as diagnostic methods using blood or urine. Examples of the tumor product include tumor-related antigens, enzymes, specific proteins, metabolites, tumor genes, products of tumor genes, and tumor-suppressor 2 0 genes. In some cancers, a carcinoembryonic antigen CEA, glycoproteins CA19-9 and CA125, a prostate-specific antigen PSA, calcitonin which is a peptide hormone produced in thyroid and the like are utilized as tumor markers in cancer diagnosis. However, in most types of cancers, there are no tumor markers useful for cancer diagnosis. Further, since most of the tumor markers currently known exist only in 2 5 very small amounts (e.g., in the order of pg/mL) in body fluid, their detection requires a highly sensitive measurement method or a special technique. Under such circumstances, if a novel cancer test method by which various cancers can be

2017239516 04 Oct 2017 detected by simple operations is provided, its use for diagnosis of various cancers are expected to be developed.

[0004]

The method would be very useful if it further enables diagnosis of cancers developed in invisible parts, assessment of the stage of cancer progression, assessment of the grade of cancer malignancy, follow-up of postoperative patients, diagnosis of recurrence, diagnosis of metastasis, monitoring of therapy and the like, in addition to detection of cancers, [0005]

More particularly, if a method which enables diagnosis of cancers developed in invisible parts is provided, it would be useful for early detection of cancers in parts where cancers are hardly realized, for example, the inside of the abdominal cavity. Further, even in cases where the tumor is too small to be found visually, detection of such cancers that cannot be found by ultrasonography, CT (computed tomography) or 15 MRI (magnetic resonance imaging) is also made possible.

[0006]

The stage of cancer progression is classified based on the extent of expansion of the tumor at the primary site and whether or not metastasis has occurred to a regional lymph node or a distant organ. In general, the disease stage is classified 2 0 into 5 stages, wherein a larger number indicates a more advanced state. Although, strictly speaking, the definition varies depending on organs, the disease stage 0 indicates a cancer staying within epithelium and the stage IV indicates a cancer with distant metastasis. If the stage of cancer progression as described above can be determined, determination of an appropriate therapeutic strategy and, in addition, 2 5 assessment of therapeutic effects of anticancer drugs are made possible. In regard to the determination of the therapeutic strategy, for example, some of prostate cancers are low malignant and hardly progress, hence do not require treatment; and others are

2017239516 04 Oct 2017 progressive and metastasize to bones and/or the like, causing pain and death of patients. Since hormonotherapy and extirpative surgery are accompanied by side effects, it is necessary to appropriately judge and determine the therapeutic approach. Further, if whether the selected anticancer drug is appropriate or not, when to end the 5 administration of the anticancer drug and the like can be appropriately judged, the physical and economical burden on the patient can also be reduced. Therefore, it is important that the stage of progression can be assessed.

[0007]

One of the characteristics of cancer cells is blastogenesis, that is, dedifferentiation. Except for a part of cancers, lower-differentiated cancer cells such as those poorly differentiated or undifferentiated grow more rapidly after the metastasis, and the prognosis is poor. Such cancers are said to be highly malignant. Conversely, highly-differentiated cancer cells, that is, those showing a high degree of cell differentiation maintain the structural and functional traits of the organ from which they were originated, and can be said to be less malignant. If such cancer malignancy can be determined, it is made possible to secure a larger margin of excision of the tumor in cases where its malignancy is high even if the size of the tumor is small, as well as to follow up the patient, paying attention to larger areas in the surrounding tissues.

[0008]

In cases where diagnosis of the postoperative course including recurrence and metastasis is possible, diagnosis of whether the tumor has been completely excised by the operation is made possible. Since recurrence is likely to occur in cases where the excision was incomplete, this can be used as a basis forjudging whether a more

5 careful follow up of the patient at short intervals is necessary and, in some cases, for deciding whether an early reoperation should be carried out. Furthermore, recurrent cancer can be found at its early stage with a high possibility. In the case of distant

2017239516 04 Oct 2017 metastasis, its detection is likely to be late, but if a method which enables diagnosis of metastasis is provided, a basis for deciding whether the region to be checked should be extended in addition to the site where the tumor was excised and the vicinity thereof can be obtained.

[0009]

If monitoring of therapy is possible, an appropriate therapeutic method or combination of therapeutic methods can be selected among various therapeutic methods to optimize the therapy. If one can see the therapeutic effects of anticancer agents, selection of the dosing periods and the types and doses of anticancer agents may be made easier. Further, after excision of the tumor, one can know presence/absence of remaining tumor, and during follow up of the patient, one can have a clue to find metastasis or recurrence as early as possible, so that initiation of early treatment is possible. If monitoring of a therapeutic effect is possible, whether the therapeutic approach was appropriate and whether the therapeutic approach should be changed to another can be judged.

[0010]

It is known that dogs age 7 times faster than human. Recently, a companion animal is kept as a member of a family and often has a lifestyle similar to that of its owner. Therefore, if the companion animal is suffering from cancer, it is possible to 2 0 predict that the owner has a high risk of development of cancer in the future. If accurate diagnosis of cancer in companion animals is possible, it is expected to be useful as a clue for prophylaxis of cancer in the owners.

[0011]

It is said that about 6,390,000 and about 17,640,000 dogs are currently kept in

5 Japan and the U.S., respectively. Since, in addition to rabies vaccination, combination vaccines such as pentavalent, heptavalent and octavalent vaccines have become popular, occurrence of highly lethal infectious diseases such as canine

2017239516 04 Oct 2017 parvovirus infection, canine distemper virus infection, canine parainfluenza (kennel cough), canine adenovirus type II infection (kennel cough), canine infectious hepatitis, canine coronavirus infection and leptospirosis decreased. Therefore, the average life expectancy of dogs increased, and aged dogs of 7 years old or older occupy 35.5% of the total number of the kept dogs. As the causes of death, cancer, hypertension, cardiac diseases and the like are consistently increasing like in human. In the U.S., about 4,000,000 dogs/year are diagnosed as cancer, and it is also said that about 1,600,000 dogs in Japan potentially have a certain tumor.

[0012]

However, there exist no simple diagnostic agents for animal cancers so far, and in the field of animal healthcare, test methods such as photography by X-ray, CT and MRI are not commonly used. Their diagnosis is carried out by palpation, simple blood test and a test by radiography, which largely depend on experience of a veterinarian. Although some veterinarians have begun to employ a test method using serum, human tumor markers are used in the method since canine tumor markers have not been found yet.

[0013]

Accurate diagnosis of cancer requires an abdominal operation, and there are large problems of the physical burden to a dog and the economical burden to its

0 owner. If diagnosis of cancers can be conveniently carried out in companion animals such as dogs and cats, early detection and accurate diagnosis of the cancers are made possible, which is useful for treatment of the cancers in the companion animals. Further, if a method which enables such simple diagnosis of cancers using serum is provided, it is expected that the method not only makes it possible to

5 diagnose cancers but also contributes a great deal to periodic health examination, preoperative diagnosis and determination of the therapeutic strategy.

[0014]

2017239516 04 Oct 2017

Unlike in human, medical examination is not popular in companion animals.

Therefore, in many cases, tumor in a companion animal is found at its late stage, and the owner realizes the tumor and takes his animal only after the tumor has grown bigger. In cases where the grown tumor is malignant, surgical therapy such as operation and administration of an anticancer agent or the like are very often too late.

Therefore, in cases where the cancer was judged as malignant by a veterinarian, anticancer drug therapy is generally carried out without operation. Even in cases where an operation is carried out, the operation must be strictly controlled to secure the margin and prevent dispersion of blood and cells during the operation. It is 10 desirable to start anticancer drug therapy immediately after the operation and to follow up the patient at short intervals. It is expected that early detection of cancers is made easier if the above-described diagnosis is adopted into the medical examination of dogs, so-called dog dock, which is recently being popularized.

[0015]

On the other hand, in the case of a benign tumor, an operation can be decided to be carried out even if the tumor is large. All that is required is to care the excised area, and there is no need for treatment with neither expensive anticancer drugs nor being nervous about the follow up.

[0016]

In view of the above-described circumstances, if a simple means for detecting cancers with a high sensitivity which can be applied to cancer diagnosis in animals is provided, accurate and efficient therapy is made possible, which is highly advantageous to both owners and veterinarians.

[0017]

5 Non-patent Literature 1: Investigation by Ministry of Health, Labour and Welfare,

2004

Non-patent Literature 2: Nikkei Science, 2007, March, pp. 80-88

2017239516 04 Oct 2017

Non-patent Literature 3: Clinical Tests, 2003, December, vol. 47, No. 13, p. 1641 1654

Non-patent Literature 4: Statistics of Diseases of Dogs and Cats, 2005, January

Non-patent Literature 5: Companion Animal Health Products: 2006 Edition By Tim 5 Wesley, ANIMAL PHARM REPORTS

Non-patent Literature 6: Expansion of Cancer and Stage of Progression. Hideaki Tsukuma, Department of Cancer Control and Statistics, Osaka Medical Center for Cancer and Cardiovascular Diseases

Non-patent Literature 7: Proteins, Nucleic Acids and Enzymes, vol. 50, No. 11, p.

1405-1412

Non-patent Literature 8: J Cell Sci. 115: 1825-35

Non-patent Literature 9: Blood. 95: 1788-96

Non-patent Literature 10: Mol Endocrinol. 9: 243-54 (1995)

Non-patent Literature 11: J Cell Biol. 145: 83-98 (1999)

DISCLOSURE OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION [0018]

An object of the present invention is to provide means for detecting a cancer(s) which is useful in diagnosing a cancer(s).

0 MEANS FOR SOLVING THE PROBLEM [0019]

The present inventors intensively studied to obtain a cDNA encoding a protein which binds to an antibody existing in serum derived from a tumor-bearing living body by the SEREX method using a cDNA library derived from canine testis 2 5 and serum of a tumor-bearing dog, which cDNA was used to prepare a polypeptide having the amino acid sequence shown in SEQ ID NO:2, a canine calmegin protein having the amino acid sequence shown in SEQ ID NO: 16, a canine centrosomal

2017239516 04 Oct 2017 protein (which may be hereinafter abbreviated as CEP) having the amino acid sequence shown in SEQ ID NO :26, and the canine thyroid hormone receptor interactor 11 (which may be hereinafter described as TRIP11) having the amino acid sequence shown in SEQ ID NO:45. Further, based on a human gene homologous to the obtained gene, a polypeptide having the amino acid sequence shown in SEQ ID NO :4, a human calmegin protein having the amino acid sequence shown in SEQ ID NO: 18, a human CEP having the amino acid sequence shown in SEQ ID NO;28, and a human TRIP11 having the amino acid sequence shown in SEQ ID NO:47 were prepared. The inventors found that genes encoding these proteins are specifically expressed in canine and human testis and malignant cancer cells (see, Examples A-l, B-l, C-l and D-l), and that recombinant proteins prepared based on the amino acid sequences of these proteins specifically react with the serum in cancer-bearing living body, as well as that each of the above-mentioned polypeptides and homologous factors thereof can be specifically detected in a cancer-bearing living body by an antibody prepared by using the respective recombinant proteins, thereby completing the present invention.

[0020]

That is, the present invention provides a method for detecting a cancer(s), which is applied to a sample separated from a living body and comprises measuring an expression of at least one of the polypeptides (a) to (d) below:

(a) a polypeptide produced in said living body and having a reactivity to bind to an antibody against a polypeptide having the amino acid sequence shown in SEQ ID NO :2 or 4 by antigen-antibody reaction.;

(b) calmegin;

(c) a polypeptide having a reactivity to bind to an antibody against a centrosomal protein having the amino acid sequence shown in SEQ ID NO:26, 28 or 42 by antigen-antibody reaction;

2017239516 04 Oct 2017 (d) thyroid hormone receptor interactor 11.

[0021]

The present invention also provides a reagent for detecting a cancer(s), comprising a polypeptide which immunologically reacts with an antibody induced in a living body against any one of the polypeptides (i) to (1) below:

(i) a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or 4;

(j) calmegin;

(k) a centrosomal protein having the amino acid sequence shown in SEQ

ID NO:26,28 or 42;

(l) thyroid hormone receptor interactor 11.

[0022]

The present invention further provides a reagent for detecting a cancer(s), comprising an antibody which immunologically reacts with any one of the polypeptides (m) to (p) below or antigen-binding fragment thereof:

(m) a polypeptide produced in a living body and having a reactivity to bind to an antibody against a polypeptide having the amino acid sequence shown in SEQ ID NO :2 or 4 by antigen-antibody reaction;

(n) calmegin;

(o) a polypeptide produced in a living body and having a reactivity to bind to an antibody against a centrosomal protein having the amino acid sequence shown in SEQ ID NO:26,28 or 42 by antigen-antibody reaction;

(p) thyroid hormone receptor interactor 11.

[0023]

5 The present invention still further provides a reagent for detecting a cancer(s), comprising a polynucleotide which specifically hybridizes with a partial region of the base sequence shown in any one of SEQ ID NOs:l, 3, 15, 17, 25,27, 41,44 and 46 in ίο

2017239516 04 Oct 2017

SEQUENCE LISTING.

EFFECTS OF THE INVENTION [0024]

By the present invention, a novel method for detecting a cancer(s) was provided. As will be concretely described in Examples below, recombinant polypeptides prepared based on the amino acid sequence shown in SEQ ID NO:2 or 4, the amino acid sequence of calmegin, the amino acid sequence of CEP shown in SEQ ID NO:26,28 or 42 and the amino acid sequence of TRIP 11 react with antibodies which specifically exist in serum of cancer patients. Therefore, cancers in a living body can be detected by measuring the antibody in a sample according to the method of the present invention. Cancers in a living body can also be detected by measuring the antigen protein per se which the antibody recognizes. Because the method of the present invention makes it possible to detect invisible small cancers and cancers which exist in a deep part of a body, it is also useful for early detection of cancers in medical examinations and the like. If the method of the present invention is used in following-up of the patients after cancer therapy, recurrence of the cancer can be detected in its early stage. Moreover, the method of the present invention makes it possible to assess the stage of cancer progression such as growth of the tumor, invasion of the tumor to the surrounding tissues, and metastasis of the

0 cancer to lymph nodes and distant organs. Furthermore, the method of the present invention makes it possible to assess the grade of cancer malignancy, because patients suffering from more malignant cancer have more amount of the antibody mentioned above in serum compared to those suffering from less malignant cancer. Furthermore, based on the increase or decrease of the above-mentioned antibody in

5 serum, how much the administered anticancer drug is effective, or whether a portion of the tumor is left in the patient after extirpation of the tumor can be assessed, as well as a clue to find metastasis and/or recurrence as early as possible can be

2017239516 04 Oct 2017 obtained during the follow-up. Thus, by the method of the present invention, monitoring of the therapy, which provides a basis for adoption of the therapeutic approach, such as whether the therapeutic approach applied to a patient is appropriate or not, whether the approach should be changed to another or not, or whether any 5 therapy should be started or not, can be attained. Furthermore, as shown in the

Examples below, mRNAs encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or 4, calmegin, CEP having the amino acid sequence shown in SEQ ID NO:26,28 or 42, and encoding TRIP11 are highly expressed specifically in the testis and cancer cells. Therefore, cancers can be detected by measuring the 10 mRNAs.

BRIEF DESCRIPTION OF THE DRAWINGS [0025]

Fig. 1 shows the expression pattern of the gene identified in Example A-l in normal tissues and tumor cell lines. Reference numeral 1: the expression pattern of 15 the identified gene; Reference numeral 2: the expression pattern of the GAPDH gene.

Fig. 2 shows the detection by Coomassie staining of the recombinant canine protein, which is an example of the polypeptide used in the present invention, produced in E. coli and purified in Example A. Reference numeral 3: the band for the recombinant canine protein.

0 Fig. 3 shows some of the results of cancer diagnosis in cancer-bearing dogs carried out using the recombinant canine protein prepared in Example A.

Fig. 4 shows some of the results of detailed cancer diagnosis in cancerbearing dogs carried out using the recombinant canine protein prepared in Example A.

Fig. 5 shows the expression pattern of the gene encoding calmegin protein in

5 normal tissues and tumor cell lines. Reference numeral 1: the expression pattern of the gene encoding calmegin protein; Reference numeral 2: the expression pattern of the GAPDH gene.

2017239516 04 Oct 2017

Fig. 6 shows the detection by Coomassie staining of canine calmegin, which is an example of the polypeptide used in the present invention, produced in E. coli and purified in Example B. Reference numeral 3: the band for the canine calmegin protein.

Fig. 7 shows some of the results of cancer diagnosis in cancer-bearing dogs carried out using the canine calmegin protein prepared in Example B.

Fig. 8 shows some of the results of detailed cancer diagnosis in cancerbearing dogs carried out using the canine calmegin protein prepared in Example B.

Fig. 9 shows the expression pattern of the gene encoding CEP in normal tissues and tumor cell lines. Reference numeral 1: the expression pattern of the gene encoding CEP; Reference numeral 2: the expression pattern of the GAPDH gene.

Fig. 10 shows the detection by Coomassie staining of the polypeptide derived from canine CEP, which is an example of the polypeptide used in the present invention, produced in E. coli and purified in Example C. Reference numeral 3: the band for the polypeptide derived from canine CEP.

Fig. 11 shows some of the results of cancer diagnosis in cancer-bearing dogs carried out using the polypeptide derived from canine CEP prepared in Example C.

Fig. 12 shows some of the results of detailed cancer diagnosis in cancer2 0 bearing dogs carried out using the polypeptide derived from canine CEP prepared in Example C.

Fig. 13 shows the expression pattern of the gene encoding the TRIP11 protein in normal tissues and tumor cell lines. Reference numeral 1: the expression pattern of the gene encoding the TRIP11 protein; Reference numeral 2: the expression

5 pattern of the GAPDH gene.

Fig. 14 shows the detection by Coomassie staining of the polypeptide derived from canine TRIP11, which is an example of the polypeptide used in the present

2017239516 04 Oct 2017 invention, produced in E. coli and purified in Example D. Reference numeral 3: the band for the polypeptide derived from canine TRIP11.

Fig. 15 shows some of the results of cancer diagnosis in cancer-bearing dogs carried out using the polypeptide derived from canine TRIP11 prepared in Example

D. '

Fig. 16 shows some of the results of detailed cancer diagnosis in cancerbearing dogs carried out using the polypeptide derived from canine TRIP 11 prepared in Example D.

BEST MODE FOR CARRYING OUT THE INVENTION [0026]

In the method of the present invention, the expression of a prescribed polypeptide is measured using a sample separated from a living body. The method for measuring the expression of a polypeptide using the sample includes a method in which an antibody against the polypeptide, which antibody is contained in the sample, 15 is measured by immunoassay (Method 1); a method in which the polypeptide per se contained in the sample is measured by immunoassay (Method 2); and a method in which mRNA contained in the sample which encodes the polypeptide is measured (Method 3). In the method of the present invention, the expression of the polypeptide may be measured by any of these three methods. In the present

0 invention, the term measurement includes detection, quantification and semiquantification.

[0027]

The above-mentioned prescribed polypeptide whose expression is measured in the method of the present invention is at least one of the polypeptides (a) to (d)

5 below:

(a) a polypeptide produced in the living body and having a reactivity to bind to an antibody against a polypeptide having the amino acid sequence shown in

2017239516 04 Oct 2017

SEQ ID NO:2 or 4 by antigen-antibody reaction;

(b) calmegin;

(c) a polypeptide having a reactivity to bind to an antibody against a centrosomal protein having the amino acid sequence shown in SEQ ID NO:26, 28 or

42 by antigen-antibody reaction;

(d) thyroid hormone receptor interactor 11.

[0028]

As shown in the following Examples, cancers are successfully detected even by measuring the expression of just one of these polypeptides. Therefore, in the 10 present invention, the expression of just one of the polypeptides (a) to (d) may be measured, as well as two or more of the polypeptides (a) to (d) may be measured in combination. When two or more polypeptides are measured, cancers can be detected with higher accuracy (see, Example E below).

[0029]

The polypeptide (a) is a polypeptide which is produced in a living body and has a reactivity to bind to an antibody against a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or 4 by antigen-antibody reaction. In other words, the prescribed polypeptide whose expression should be measured is a polypeptide which has the same antigenicity as a canine-derived polypeptide of SEQ ID NO:2 or 20 as a human-derived polypeptide of SEQ ID NO:4.

[0030]

Specific examples of such a polypeptide include a canine-derived polypeptide of SEQ ID NO:2 and a human-derived polypeptide of SEQ ID NO:4. These polypeptides are the very corresponding antigen of an antibody against a polypeptide 2 5 having the amino acid sequence shown in SEQ ID NO:2 or 4, and thus included in the above-mentioned prescribed polypeptide. Specific examples of the polypeptide also include a polypeptide which is derived from other mammals and has the same

2017239516 04 Oct 2017 antigenicity as the above-mentioned canine- or human-derived polypeptide (such a polypeptide is hereinafter referred to as homologous factor, and the human-derived polypeptide as described above may also be referred to as human homologous factor of the canine-derived polypeptide).

[0031]

SEQ ID NO:2 shows the amino acid sequence of the polypeptide with unknown function identified in the Examples below as a polypeptide which binds to an antibody existing specifically in serum derived from a cancer-bearing dog (the antibody may also be hereinafter referred to as cancer-specific antibody of dogs), 10 which identification was carried out by the SEREX method using a canine testisderived cDNA library and serum of cancer-bearing dogs (see, Example A-l). Therefore, cancers in dogs can be detected by measuring this cancer-specific antibody against a polypeptide of SEQ ID NO:2 in accordance with Method 1 above (see, Examples A-3 and A-4). Cancers in dogs can also be detected by measuring 15 the polypeptide of SEQ ID NO:2 per se, which is the antigen, in accordance with Method 2 above (see, Examples A-5 and A-6). Moreover, since the expression of mRNA encoding the antigen polypeptide is significantly high in the testis and cancer cells as shown in the following Examples (see, Example A-l), cancers in dogs can also be detected by measuring the mRNA. It is noted that, although the amino acid 2 0 sequence of the canine polypeptide shown in SEQ ID NO:2 is registered in the NCBI database under the Accession No. XP_535343 (protein) and Accession No. XM_535343 (coding gene), its function has not been reported yet.

[0032]

SEQ ID NO:4 shows the amino acid sequence of the human homologous

5 factor of the canine-derived polypeptide described above, which was found by BLAST homology search. The base sequence encoding the human homologous factor and the amino acid sequence thereof are shown in SEQ ID NOs:3 and 4,

2017239516 04 Oct 2017 respectively, and also registered in the NCBI database under Accession No. NP_689873 (protein) and Accession No, NM_152660 (coding gene). Similarly to the canine-derived polypeptide described above, any functions of the human homologous factor have not been reported. As concretely shown in the following

Examples, similarly to the canine-derived polypeptide of SEQ ID NO:2, the expression level of mRNA encoding the human homologous factor is significantly high in human testis and cancer cells, and an antibody against the human homologous factor is not detected in healthy humans. Therefore, cancers in humans can be detected by determining the expression of a polypeptide of SEQ ID NO:4 in the humans.

[0033]

Specific examples of the homologous factor in other mammals which has the same antigenicity as the above-mentioned canine-derived polypeptide or the human homologous factor thereof include, for example, the polypeptide which specifically exists in cancer-bearing cats as shown in the following Examples. This feline polypeptide immunologically reacts with not only an antibody prepared by using as an immunogen the canine-derived polypeptide of SEQ ID NO:2, but also an antibody prepared by using as an immunogen the human homologous factor of SEQ ID NO:4 (see, Examples A-5 and A-6). Therefore, this feline polypeptide is the feline

0 homologous factor which has the same antigenicity as the above-described canineand human-derived polypeptides, and included in the scope of a polypeptide which has a reactivity to bind to an antibody against a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or 4 by antigen-antibody reaction, whose expression is to be measured in the present invention. As concretely described in

5 the Examples below, an antibody induced in cats against the feline homologous factor is detected only in cancer-bearing cats and not detected in healthy cats. The feline homologous factor per se, which is the antigen, is also detected only in cancer17

2017239516 04 Oct 2017 bearing cats and not detected in healthy cats. Therefore, by measuring the expression of the homologous factor in mammals other than dogs and humans, cancers in the mammals can be detected.

[0034]

The polypeptide (a) described above is preferably a polypeptide having the amino acid sequence shown in SEQ ID NO:2 in SEQUENCE LISTING, or a polypeptide which has a homology of not less than 95% thereto and is produced in a living body. The homology between the canine-derived polypeptide (SEQ ID NO:2) and the human homologous factor thereof (SEQ ID NO:4) is 93% in terms of 10 base sequence and 99% in terms of amino acid sequence. Although dogs and humans are genetically distant, the homologous factor in such a genetically distant species shares a very high homology of 99% at amino acid level. Therefore, it is believed that the homologous factor in mammals other than human also shares as high homology as not less than 95% with the canine-derived polypeptide of SEQ ID 15 NO:2.

[0035]

The above-described polypeptide (b), Calmegin, was identified as a protein which is expressed specifically at the time of differentiation of a spermatid, and has a chaperone activity in vitro. Since it is expressed only in testis and disappears in a 2 0 mature sperm, calmegin is considered to have a function to fold proteins involved in differentiation of spermatid (Non-patent Literature 7: Naokazu Inoue, Ryo Yamaguchi and Masahito Ikawa, Protein, Nucleic Acid and Enzyme, vol. 50, No. 11, 1405-1412). However, there has been no report showing that the protein is expressed in a cancer and useful for cancer diagnosis and the like.

[0036]

SEQ ID NO: 16 shows the amino acid sequence of canine calmegin. The canine calmegin having this amino acid sequence was identified as a polypeptide

2017239516 04 Oct 2017 which binds to an antibody existing specifically in serum derived from a cancerbearing dog, which identification was carried out by the SEREX method using a canine testis-derived cDNA library and serum of cancer-bearing dogs (see, Example B-1). That is, in cancer-bearing dogs, an antibody against calmegin having the amino acid sequence shown in SEQ ID NO: 16 is specifically induced. Therefore, cancers in dogs can be detected by measuring the above-mentioned antibody against calmegin having the amino acid sequence shown in SEQ ID NO: 16 in accordance with Method 1 above (see, Examples B-3 and B-4). Cancers in dogs can also be detected by measuring calmegin of SEQ ID NO: 16 per se, which is the antigen, in accordance with Method 2 above (see, Examples B-5 and B-6). Moreover, since the expression of mRNA encoding calmegin is significantly high in the testis and cancer cells as shown in the following Examples (see, Example B-l), cancers in dogs can also be detected by measuring the mRNA.

[0037]

In the method of the present invention, not only the canine calmegin of SEQ

ID NO: 16 but also calmegin in other mammals (hereinafter also referred to as homologous factor of canine calmegin; in cases where the simple term calmegin is used, not only canine calmegin but also other mammalian calmegin are referred to by the term) may be measured. As concretely described in the Example below,

0 similarly to canine calmegin of SEQ ID NO: 16, the expression level of mRNA encoding human calmegin is also significantly high in human testis and cancer cells, and an antibody against human calmegin is not detected in healthy humans. An antibody against feline calmegin is detected only in cancer-bearing cats and not detected in healthy cats. Therefore, by measuring the expression of calmegin in

5 mammals other than dogs, cancers in the mammals can also be detected. Besides canine calmegin, examples of the calmegin to be measured in the method of the present invention include, but not limited to, human calmegin, feline calmegin and

2017239516 04 Oct 2017 the like. The base sequence encoding human calmegin and the amino acid sequence thereof are shown in SEQ ID NOs:17 and 18 in SEQUENCE LISTING, respectively, and the homology between human calmegin and canine calmegin is 90% in terms of base sequence and 89% in terms of amino acid sequence. Although dogs and humans are genetically distant, the calmegin in such genetically distant species share a very high homology of 89% at amino acid level. Therefore, it is believed that calmegin in mammals other than human also shares as high homology as not less than about 80% with canine calmegin. That is, calmegin to be measured in the method of the present invention preferably has a homology of not less than 80%, more preferably not less than 85% to canine calmegin shown in SEQ ID NO: 16, although not restricted thereto.

[0038]

The polypeptide (c) described above is a polypeptide which has a reactivity to bind to an antibody against centrosomal protein (CEP) having the amino acid sequence shown in SEQ ID NO:26,28 or 42 by antigen-antibody reaction. In other words, the polypeptide (c) described above is a polypeptide which has the same antigenicity as canine-derived CEP of SEQ ID NO :26 or 42 or human-derived CEP of SEQ ID NO:28.

[0039]

0 Specific examples of such a prescribed CEP include canine-derived CEP of

SEQ ID NO :26 or 42, and human-derived CEP of SEQ ID NO :28. These CEPs are the very corresponding antigen of an antibody against CEP having the amino acid sequence shown in SEQ ID NO:26, 28 or 42, and thus included in a prescribed CEP as mentioned above. Specific examples of the CEP also include CEP derived from

5 other mammals which has the same antigenicity as the above-mentioned canine- or human-derived CEP (such CEP is hereinafter referred to as homologous factor, and human-derived CEP mentioned above may also be referred to as human

2017239516 04 Oct 2017 homologous factor of canine-derived CEP).

[0040]

SEQ ID NO:26 shows the amino acid sequence of canine CEP identified as a polypeptide which binds to an antibody existing specifically in serum derived from a 5 cancer-bearing dog (hereinafter also referred to as a canine cancer-specific antibody), which identification was carried out by the SEREX method using a canine testis-derived cDNA library and serum of cancer-bearing dogs (see, Example C-l). Therefore, cancers in dogs can be detected by measuring the abovementioned antibody against canine CEP having the amino acid sequence shown in 10 SEQ ID NO:26 in accordance with Method 1 above (see, Examples C-3 and C-4).

Cancers in dogs can also be detected by measuring CEP of SEQ ID NO :26 per se, which is the antigen, in accordance with Method 2 above (see, Examples C-5 and C6). Moreover, since the expression of mRNA encoding CEP of SEQ ID NO:26 is significantly high in the testis and cancer cells as shown in the following Examples 15 (see, Example C-l), cancers in dogs can also be detected by measuring the mRNA.

CEP is a protein which is required by the centrosome to control microtubules, and also involved in maturation of the centrosome. Frequent occurrence of chromosomal translocation is known in a part of myeloproliferative disorders, and since the CEP gene exists at the point where the translocation occurs, it is considered 20 to have a certain relationship with the disorders. However, there has been no report showing that the protein is expressed in a cancer and useful for cancer diagnosis (Non-patent Literature 8: J Cell Sci. 115:1825-35; Non-patent Literature 9: Blood. 95:1788-96).

[0041]

5 SEQ ID NO:42 shows the amino acid sequence of a known canine CEP registered in a database, which was found as a protein sharing very high homology with the obtained canine CEP mentioned above by BLAST search (see, Example C21

2017239516 04 Oct 2017

1). The base sequence of this known canine CEP is shown in SEQ ID NO:41. Similarly to canine CEP of SEQ ID NO:26, canine CEP of SEQ ID NO:42 is also considered to be highly expressed in cancer-bearing dogs, and cancers in dogs can be detected by determining the expression of this known canine CEP as concretely described in the following Examples.

[0042]

SEQ ID NO:28 shows the amino acid sequence of a human homologous factor of the canine-derived CEP mentioned above, which amino acid sequence was found by BLAST homology search. The base sequence encoding the human 10 homologous factor and the amino acid sequence thereof are shown in SEQ ID NOs:27 and 28 in SEQUENCE LISTING, respectively. As concretely described in the Examples below, similarly to canine-derived CEP of SEQ ID NO:26, the expression level of mRNA encoding the human homologous factor is significantly high in human testis and cancer cells, and an antibody against the human homologous 15 factor is not detected in healthy humans. Therefore, cancers in humans can be detected by determining the expression of CEP of SEQ ID NO:28 in the humans. [0043]

Specific examples of the homologous factor in other mammals which has the same antigenicity as the above-described canine-derived CEP or as the human

0 homologous factor thereof include, for example, CEP which specifically exists in cancer-bearing cats as shown in the following Examples. The feline CEP immunologically reacts with not only an antibody prepared by using as an immunogen canine-derived CEP of SEQ ID NO:26 or 42, but also an antibody prepared by using as an immunogen the human homologous factor of SEQ ID NO:28

5 (see, Examples C-5 and C-6). Therefore, this feline CEP is a feline homologous factor which has the same antigenicity as the above-mentioned canine- and humanderived CEPs, and thus included in the scope of a CEP which has a reactivity to

2017239516 04 Oct 2017 bind to an antibody against CEP having the amino acid sequence shown in SEQ ID NO:26,28 or 42 by antigen-antibody reaction, whose expression is to be measured in the present invention. As concretely described in the Examples below, an antibody induced in cats against the feline homologous factor is detected only in cancer-bearing cats and not detected in healthy cats. The feline homologous factor per se, which is the antigen, is also detected only in cancer-bearing cats and not detected in healthy cats. Therefore, by measuring the homologous factor in mammals other than dogs and humans, cancers in the mammals can also be detected. [0044]

Preferably, the CEP whose expression should be measured in the detection method of the present invention is CEP having the amino acid sequence shown in SEQ ID NO:26 or 42 in SEQUENCE LISTING, or a polypeptide which has a homology of not less than 80% thereto and is produced in a living body. The homology between canine-derived CEP and human homologous factor thereof is

87% in terms of base sequence and 84% in terms of amino acid sequence.

Although dogs and humans are genetically distant, the homologous factor in such a genetically distant species shares very high homology of 84% at amino acid level. Therefore, it is believed that the homologous factor in mammals other than human also shares as high homology as not less than 80% with the canine CEP.

[0045]

The above-described polypeptide (d), TRIP11 (thyroid hormone receptor interactor 11), was first identified as a factor which interacts with the thyroid hormone receptor β, and its binding to Golgi bodies and microtubules also became evident, so that it is considered to play a role in maintaining the shapes of these

5 organelles by making links between Golgi bodies, microtubules and the like. However, there has been no report showing that the protein is expressed in a cancer and useful for cancer diagnosis and the like (Non-patent Literature 10: Mol

2017239516 04 Oct 2017

Endocrinol. 9:243-54 (1995); Non-patent Literature 11: J Cell Biol. 145: 83-98 (1999)).

[0046]

SEQ ID NO:45 shows the amino acid sequence of canine TRIP 11. The canine TRIP11 having this amino acid sequence was identified as a polypeptide which binds to an antibody existing specifically in serum derived from a cancerbearing dog, which identification was carried out by the SEREX method using a canine testis-derived cDNA library and serum of cancer-bearing dogs (see, Example D-l). That is, in cancer-bearing dogs, an antibody against TRIP11 having the amino acid sequence shown in SEQ ID NO:45 is specifically induced. Therefore, cancers in dogs can be detected by measuring the above-mentioned antibody against TRIP11 having the amino acid sequence shown in SEQ ID NO:45 in accordance with Method 1 above (see, Examples D-3 and D-4). Cancers in dogs can also be detected by measuring TRIP 11 of SEQ ID NO :45 per se, which is the antigen, in accordance with

Method 2 above (see, Examples D-5 and D-6). Moreover, since the expression of mRNA encoding TRIP11 is significantly high in the testis and cancer cells as shown in the following Examples (see, Example D-l), cancers in dogs can also be detected by measuring the mRNA.

[0047]

In the method of the present invention, not only canine TRIP 11 of SEQ ID

NO:45 but also TRIP11 in other mammals (hereinafter also referred to as homologous factor of canine TRIP11; in cases where the simple term TRIP11 is used, not only canine TRIP11 but also other mammalian TRIP11 are referred to by the term) may be measured. As concretely described in the Example below,

5 similarly to canine TRIP 11 of SEQ ID NO:45, the expression level of mRNA encoding human TRIP 11 is also significantly high in human testis and cancer cells, and an antibody against human TRIP11 is not detected in healthy humans. An

2017239516 04 Oct 2017 antibody against feline TRIP11 is detected only in cancer-bearing cats and not detected in healthy cats. Therefore, by measuring the expression of TRIP 11 in mammals other than dog, cancers in the mammals can also be detected. Besides canine TRIP 11, examples of the TRIP11 to be measured in the method of the present 5 invention include, but not limited to, human TRIP11, feline TRIPll and the like.

The base sequence encoding human TRIP 11 and the amino acid sequence thereof are shown in SEQ ID NOs:46 and 47 in SEQUENCE LISTING, respectively, and the homology between canine TRIP 11 and human TRIP11 is 88% in terms of base sequence and 86% in terms of amino acid sequence. Although dogs and humans are genetically distant, the TRIP1 Is in such genetically distant species share very high homology of 86% at amino acid level with each other. Therefore, it is believed that

TRIP 11 in mammals other than human also shares as high homology as not less than about 75% with canine TRIP11. That is, the TRIP11 whose expression should be measured in the method of the present invention preferably has a homology of not less than 75%, more preferably not less than 80% to the amino acid sequence of canine TRIP11 shown in SEQ ID NO:45, although not restricted thereto.

[0048]

It should be noted that the term having the amino acid sequence in the present invention means that amino acid residues are aligned in that order.

0 Accordingly, for example, a polypeptide having the amino acid sequence shown in SEQ ID NO:2 means a polypeptide having a size of 306 amino acid residues, whose amino acid sequence is Met Ala Ala Leu ... (snip)... He Thr Ser Pro as shown in SEQ ID NO:2. Further, a polypeptide having the amino acid sequence shown in SEQ ID NO:2 may be abbreviated as a polypeptide of SEQ ID NO:2. This also

5 applies to the term having the base sequence. It should be noted that the term polypeptide in the present invention means a molecule formed by peptide bonding of a plurality of amino acids, and includes not only polypeptide molecules having a

2017239516 04 Oct 2017 large number of amino acids constituting them, but also low molecular weight molecules having a small number of amino acids (oligopeptides) and full-length proteins. Thus, in the present invention, proteins consisting of the full length of SEQ ID NO:2,4, 16,18, 26, 28, 42,45 or 47 are also included in polypeptide.

[0049]

In Method 1 above, measurement of the cancer-specific antibody which may exist in the sample can be easily carried out by immunoassay using an antigenic substance which immunologically reacts with the antibody. The immunoassay per se is a conventional well-known method as explained in detail below. Examples of 10 the antigenic substance which may be used in the immunoassay include a polypeptide of SEQ ID NO:2, 16, 26, 42 or 45, which induces the antibody in cancer-bearing dogs. As antibodies have the cross-reactivity, a molecule may be bound to an antibody which is induced against another immunogen, as long as the molecule has any structure thereon which is similar to the epitope of the immunogen. For example, polypeptides having high amino acid sequence homology to each other often have epitopes with similar structures, and in such cases both polypeptides may have the same antigenicity. As concretely described in the Examples below, caninederived polypeptide of SEQ ID NO:2,16, 26, 42 or 45 immunologically reacts with not only an antibody induced in cancer-bearing dogs against the polypeptide but also an antibody induced in cancer-bearing cats against a feline homologous factor. The human homologous factor immunologically reacts with the above-described antibodies induced in cancer-bearing dogs and cats. Therefore, in Method 1 of the present invention, any mammalian homologous factors may be used as an antigen in the immunoassay.

[0050]

Antigenic substances having a large molecular weight and a complex structure, such as proteins, usually have a plurality of sites with different structures

2017239516 04 Oct 2017 on their surface. Therefore, such a large, complex antigenic substance induces a plurality of kinds of antibodies which respectively recognize each of the sites in a living body. That is, an antibody induced in a living body against an antigenic substance such as a protein is a polyclonal antibody, which is a mixture of a plurality 5 of kinds of antibodies. The cancer-specific antibodies found by the present inventors, which specifically exist in the serum from cancer-bearing living bodies and specifically bind to a polypeptide of SEQ ID NO:2,16, 26, 42 or 45 or homologous factors thereof by antigen-antibody reaction, are also a polyclonal antibody. It should be noted that, in the present invention, the term polyclonal 10 antibody means an antibody which exists in serum from a living body having an antigenic substance therein and is induced in the living body against the antigenic substance.

[0051]

In the Example A below, a polypeptide consisting of the entire region of SEQ 15 ID NO:2 and a polypeptide consisting of the entire region of SEQ ID NO:4, which is the human homologous factor, were prepared as an antigen for immunoassay of the cancer-specific antibody, and the reactivity of these polypeptides with the antibody in the serum derived from a cancer-bearing living body was confirmed. In the Example B below, a polypeptide consisting of the entire region of SEQ ID NO: 16 2 0 (canine calmegin) and a polypeptide consisting of the entire region of SEQ ID NO: 18 (human calmegin), which is the human homologous factor thereof, were prepared, and the reactivity of these polypeptides with the antibody in the serum derived from a cancer-bearing living body was confirmed. In the Example C below, a polypeptide consisting of a region of 1514th to 2339th amino acids of SEQ ID NO:26 (canine 2 5 CEP) and a polypeptide consisting of a region of 1513rd to 2325th amino acids of

SEQ ID NO :28 (human CEP) were prepared, and the reactivity of these polypeptides with the antibody in the serum derived from a cancer-bearing living body was

2017239516 04 Oct 2017 confirmed. In the Example D below, a polypeptide consisting of a region of 237th to 1023rd amino acids of SEQ ID NO:45 (canine TRIP11) and a polypeptide consisting of a region of 236th to 1023rd amino acids of SEQ ID NO:47 (human TRIP 11) were prepared, and the reactivity of these polypeptides with the antibody in the serum derived from a cancer-bearing living body was confirmed. However, since the antibodies mentioned above are polyclonal, a polypeptide consisting of the full length of SEQ ID NO:2, 16,26,42 or 45 or a homologous factor thereof of course binds to the antibody. A fragment of the polypeptide can also bind to the antibody contained in the serum from a cancer-bearing living body, since the polyclonal antibody may include antibodies which recognize the structure of the fragment. That is, not only a polypeptide consisting of the full length of SEQ ID NO:2,16, 26,42 or 45 or a homologous factor thereof but also a fragment thereof may be used in measurement of the polyclonal antibody specifically contained in the serum of a cancer-bearing living body, and is useful for detection of a cancer(s).

[0052]

Thus, a polypeptide used as an antigen for immunoassay in Method 1 of the present invention is not restricted to a polypeptide consisting of the full length of SEQ ID NO:2,16, 26,42 or 45 or a homologous factor thereof (e.g. SEQ ID NO:4, 18,28,47, etc.), and includes a polypeptide fragment which consists of not less than 20 7 consecutive, preferably not less than 10 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:2,16, 26,42 or 45 or of a homologous factor thereof, and immunologically reacts with a polyclonal antibody against a canine-derived polypeptide of SEQ ID NO:2,16,26,42 or 45 or a homologous factor thereof (the polypeptide fragment may also be hereinafter referred to as specifically-reactive

5 partial polypeptide for convenience). It should be noted that, as known in the art, a polypeptide having not less than about 7 amino acid residues can exert its antigenicity.

2017239516 04 Oct 2017 [0053]

However, in cases where the number of the amino acid residues are too small, the possibility that the antigen polypeptide may cross-react with antibodies against proteins which exist in the sample and are different from the canine-derived polypeptide of SEQ ID NO:2, 16,26,42 or 45 or the homologous factor thereof is increased. Therefore, in view of attaining a high accuracy in the immunoassay, a polypeptide fragment consisting of a large number of the amino acid residues is preferred as an antigen used in the immunoassay. For example, in the case of a polypeptide of SEQ ID NO:2 or a homologous factor thereof, it is desired that the number of the amino acid residues of the used polypeptide fragment should be preferably not less than 30, more preferably not less than 100, still more preferably not less than 200, still more preferably not less than 250. In the case of canine calmegin of SEQ ID NO: 16 or a homologous factor thereof, it is desired that the number of the amino acid residues should be preferably not less than 30, more preferably not less than 100, still more preferably not less than 200, still more preferably not less than 400, still more preferably not less than 550. In the case of canine CEP of SEQ ID NO:26 or 42 or a homologous factor thereof, it is desired that the number of the amino acid residues should be preferably not less than 30, more preferably not less than 100, still more preferably not less than 300, still more

0 preferably not less than 600, and the number of the amino acid residues may be not less than 1000, not less than 1500, or not less than 2000. In the case of canine TRIP 11 of SEQ ID NO:45 or a homologous factor thereof, it is desired that the number of the amino acid residues should be preferably not less than 30, more preferably not less than 100, still more preferably not less than 300, still more

5 preferably not less than 600, and the number of the residues may be not less than 1000, or not less than 1500.

[0054]

2017239516 04 Oct 2017

Specific examples of the polypeptide used as an antigen include the following polypeptides:

(e) a polypeptide having the amino acid sequence shown in SEQ ID NO:2 or 4;

(f) a polypeptide having the amino acid sequence shown in SEQ ID

NO: 16 or 18;

(g) a polypeptide consisting of not less than 500 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:26 and comprising not less than 500 consecutive amino acids located in the region of 1514th to 2339th amino acids of

SEQ ID NO:26, or a polypeptide consisting of not less than 500 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:28 and comprising not less than 500 consecutive amino acids located in the region of 1513rd to 2325th amino acids of SEQ ID NO:28;

(h) a polypeptide consisting of not less than 500 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:45 and comprising not less than

500 consecutive amino acids located in the region of 237th to 1023rd amino acids of SEQ ID NO:45, or a polypeptide consisting of not less than 500 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:47 and comprising not less than 500 consecutive amino acids located in the region of 236th to 1023rd amino acids of SEQ ID NO:47.

[0055]

Preferred examples of the polypeptide (g) above (a polypeptide of SEQ ID NO:26 or a fragment thereof, or a polypeptide of SEQ ID NO:28 or a fragment thereof) include a fragment which comprises a region of 1514th to 2339th amino

5 acids of the amino acid sequence shown in SEQ ID NO :26 and consists of not more than 1000 amino acids, and a fragment which comprises a region of 1513rd to 2325th amino acids of the amino acid sequence shown in SEQ ID NO :28 and consists of not

2017239516 04 Oct 2017 more than 1000 amino acids. More preferred examples thereof include a fragment consisting of a region of 1514th to 2339th amino acids (SEQ ID NO:35) of the amino acid sequence shown in SEQ ID NO:26, and a fragment consisting of a region of 1513rd to 2325th amino acids (SEQ ID NO:36) of the amino acid sequence shown in 5 SEQIDNO:28.

[0056]

Preferred examples of the polypeptide (h) above (a polypeptide of SEQ ID NO:45 or a fragment thereof, or a polypeptide of SEQ ID NO:47 or a fragment thereof) include a fragment which comprises a region of 237th to 1023rd amino acids 10 of the amino acid sequence shown in SEQ ID NO:45 and consists of not more than 1000 amino acids, and a fragment which comprises a region of 236th to 1023rd amino acids of the amino acid sequence shown in SEQ ID NO:47 and consists of not more than 1000 amino acids. More preferred examples thereof include a fragment consisting of a region of 237th to 1023rd amino acids (SEQ ID NO:54) of the amino 15 acid sequence shown in SEQ ID NO:45, and a fragment consisting of a region of 236th to 1023rd amino acid (SEQ ID NO:55) of the amino acid sequence shown in SEQ ID NO:47.

[0057]

It is well-known in the art that, in general, there are cases where a protein

0 antigen retains substantially the same antigenicity as the original even if the amino acid sequence of the protein is modified such that a small number of amino acids are substituted, deleted and/or inserted. Therefore, the polypeptides each of which has the same amino acid sequence as the polypeptide of SEQ ID NO:2, 16,26,42 or 45 or the homologous factor thereof except that a small number of amino acid residues are substituted, deleted and/or inserted, whose sequence has a homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, still more preferably not less than 98% to the sequence of the original polypeptide, and which

2017239516 04 Oct 2017 polypeptide specifically binds to a polyclonal antibody against a polypeptide having the amino acid sequence shown in SEQ ID NO:2,16, 26,42 or 45 or a homologous factor thereof by antigen-antibody reaction (the polypeptides may also be hereinafter referred to as specifically-reactive modified polypeptide for convenience), may also 5 be used for detection of cancers. Preferably, the specifically-reactive modified polypeptide has the same amino acid sequence as the polypeptide of SEQ ID NO :2, 16,26,42 or 45 or a homologous factor thereof (preferably having the sequence shown in SEQ ID NO:4, 18, 28 or 47) except that one or several amino acid residues are substituted, deleted and/or inserted.

[0058]

As used herein, the term homology of amino acid sequences means a value expressed in percentage which is calculated by aligning two amino acid sequences to be compared such that the number of matched amino acid residues is the maximum, and dividing the number of the matched amino acid residues by the number of the 15 total amino acid residues. When the above-described alignment is carried out, a gap(s) is/are inserted into one or both of the two sequences to be compared as required. Such alignment of sequences can be carried out using a well-known program such as BLAST, FAST A and CLUSTAL W. When a gap(s) is/are inserted, the above-described number of the total amino acid residues is calculated by counting 2 0 one gap as one amino acid residue. When the thus counted numbers of the total amino acid residues are different between the two sequences to be compared, homology (%) is calculated by dividing the number of matched amino acid residues by the number of total amino acid residues in the longer sequence. The 20 types of amino acids constituting the naturally occurring proteins may be classified into

5 groups each of which has similar properties, for example, into neutral amino acids with side chains having low polarity (Gly, He, Vai, Leu, Ala, Met, Pro), neutral amino acids having hydrophilic side chains (Asn, Gin, Thr, Ser, Tyr, Cys), acidic

2017239516 04 Oct 2017 amino acids (Asp, Glu), basic amino acids (Arg, Lys, His) and aromatic amino acids (Phe, Tyr, Trp). It is known that, in most cases, substitutions of amino acids within the same group do not change the properties of the polypeptides. Therefore, in cases where the amino acid residue(s) of the polypeptide of SEQ ID NO:2, 16,26,42 5 or 45 or a homologous factor thereof is/are substituted, the probability that the ability to bind to the corresponding antibody can be maintained may be made high by conducting the substitution(s) within the same group.

[0059]

The polypeptides which contain the above-described polypeptide used in the present invention as a partial sequence (i.e., the polypeptides used in the present invention which have other (poly)peptide(s) added at one or both ends thereof) and which specifically bind to a polyclonal antibody against a polypeptide of SEQ ID NO:2,16,26,42 or 45 or a homologous factor thereof (the polypeptides may also be hereinafter referred to as specifically-reactive added polypeptide for convenience) may also be used for detection of cancers.

[0060]

The above-described polypeptides used in the present invention may be prepared by chemical synthesis such as the Fmoc method (fluorenylmethyloxycarbonyl method), the tBoc method (Lbutyloxycarbonyl method) 20 or the like, or prepared by a conventional method using a commercially available peptide synthesizer. The polypeptides may also be easily prepared by a known genetic engineering method. For example, the desired polypeptides may be obtained by extracting RNAs from a tissue expressing a gene encoding a polypeptide of SEQ ID NO:2, 16, 26, 42 or 45 or a homologous factor thereof, preparing cDNA 25 of the gene by RT-PCR, inserting the full length or a desired part of the cDNA into an expression vector, and then introducing the vector into a host cell. The base sequences of cDNAs encoding the canine polypeptide of SEQ ID NO:2, canine

2017239516 04 Oct 2017 calmegin of SEQ ID NO: 16, canine CEPs of SEQ ID NOs:26 and 42, and canine TRIP 11 of SEQ ID NO:45 are shown in SEQ ID NO:1, SEQ ID NO: 15, SEQ ID NOs:25 and 41, and SEQ ID NO:44, respectively, and the base sequences of cDNAs encoding human homologous factors of the above polypeptides are shown in SEQ ID

NO:3, SEQ ID NO: 17 (human calmegin), SEQ ID NO:27 (human CEP), and SEQ ID

NO:47 (human TRIP11), respectively. Therefore, referring to these base sequences, primers used in RT-PCR may easily be designed. Further, as explained below, genes encoding a homologous factor in mammals other than human may be amplified by using primers designed in reference to the base sequences of canine polypeptides and human homologous factors. Therefore, cDNAs encoding e.g. a feline homologous factor may easily be prepared in the same manner as described above.

Extraction of RNAs, RT-PCR, insertion of cDNA into a vector, and introduction of a vector into a host cell may be performed by a well-known method as described below. Vectors and host cells which may be used are well-known, and various vectors and host cells are commercially available.

[0061]

The above-described host cells are not restricted as long as they can express the above-described polypeptide, and examples thereof include prokaryotic cells such as E. coli; and eukaryotic cells such as mammalian cultured cells including monkey kidney cells COS 1 and Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, and Xenopus laevis egg cells.

[0062]

In cases where prokaryotic cells are used as the host cells, an expression vector having the origin that enables its replication in a prokatyotic cell, a promoter, a ribosome binding site, a DNA cloning site, a terminator and the like is used as the expression vector. Examples of the expression vector for E. coli include the pUC system, pBluescriptll, pET expression system and pGEX expression system. By

2017239516 04 Oct 2017 incorporating DNA encoding the above-described polypeptide into such an expression vector and transforming prokaryotic host cells with the vector, followed by culturing the obtained transformant, the polypeptide encoded by the abovedescribed DNA can be expressed in the prokaryotic host cells. In this case, the polypeptide can also be expressed as a fusion protein with another protein. The DNA encoding the above-described polypeptide may be obtained by preparing cDNA by RT-PCR as described above, or may be synthesized by a conventional method using a commercially available nucleic acid synthesizer as explained below. It should be noted that the base sequences of cDNAs encoding polypeptides of SEQ ID 10 NOs:2,4,16, 18, 26,28,42,45 and 47 are shown in SEQ ID NOs:l, 3, 15,17,25, 27, 41,44 and 46 in SEQUENCE LISTING, respectively.

[0063]

In cases where eukaryotic cells are used as the host cells, an expression vector for eukaryotic cells having a promoter, splicing site, poly(A) addition site and the like 15 is used as the expression vector. Examples of such an expression vector include pKAl, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, the EBV vector, pRS, pcDNA3, pMSG and p¥ES2. In the same manner as described above, by incorporating DNA encoding the polypeptide used in the present invention into such an expression vector and transforming eukaryotic host cells with the vector, followed 20 by culturing the obtained transformant, the polypeptide encoded by the abovedescribed DNA can be expressed in the eukaryotic host cells. In cases where pIND/V5-His, pFLAG-CMV-2, pEGFP-Nl or pEGFP-Cl was used as the expression vector, the above-described polypeptide can be expressed as a fusion protein having various added tags such as His tag, FLAG tag, myc tag, HA tag or GFP.

[0064]

Introduction of the expression vector to the host cells can be carried out using a well-known method such as electroporation, the calcium phosphate method, the

2017239516 04 Oct 2017 liposome method or the DEAE dextran method.

[0065]

Isolation and purification of a polypeptide of interest from the host cells can be carried out by a combination of known separation operations. Examples of the 5 operations include treatment by a denaturant such as urea or by a surfactant;

ultrasonication treatment; enzyme digestion; salting-out and solvent fractional precipitation; dialysis; centrifugation; ultrafiltration; gel filtration; SDS-PAGE; isoelectric focusing; ion-exchange chromatography; hydrophobic chromatography; affinity chromatography; and reversed-phase chromatography.

[0066]

The polypeptides obtained by the above method include those in the form of a fusion protein with another arbitrary protein. Examples thereof include fusion proteins with glutathione S-transferase (GST) and with a His tag. Such a polypeptide in the form of a fusion protein is also included in the specifical ly-reactive 15 added polypeptide described above, and may be used in Method 1 of the present invention. Further, in some cases, a polypeptide expressed in a transformed cell is modified in various ways in the cell after translation thereof. Such a polypeptide having a post-translational modification may also be used in Method 1 of the present invention, as long as it has an ability to bind to a polyclonal antibody against a 2 0 polypeptide of SEQ ID NO:2 or 4. Examples of such a post-translational modification include elimination of N-terminus methionine, N-terminus acetylation, glycosylation, limited degradation by an intracellular protease, myristoylation, isoprenylation and phosphorylation.

[0067]

5 Measurement of the antibody in a sample may easily be carried out by immunoassay using the above-described polypeptide as an antigen. Immunoassays per se are well-known in the art, and includes, when classified based on the reaction

2017239516 04 Oct 2017 mode, sandwich method, competition method, agglutination method, Western blot method and the like. When classified based on the label, immunoassays include radioimmunoassay, fluorescence immunoassay, enzyme immunoassay, biotin immunoassay and the like, and the immunoassay of the above-described antibody may be carried out by any of these immunoassays. Although not restricted, the sandwich ELISA and competition method may be preferably used as an immunoassay of the above antibody in the present invention, as these methods are simple and do not require a large-scale apparatus. In cases where enzymes are used as a label of antibodies, the used enzyme is not particularly restricted as long as it satisfies such conditions that the turnover number is large, that the enzyme is stable even when it is bound to an antibody, that it specifically colors its substrate and the like. For example, enzymes used in an ordinary enzyme immunoassay such as peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase may be used. Enzyme inhibitors, coenzymes and the like may also be used. Binding of these enzymes with an antibody may be carried out by a known method using a cross-linking agent such as a maleimide compound. As a substrate, known substances may be used depending on the kind of the used enzyme. For example, in cases where peroxidase is used as an enzyme, 3,3’,5,5’-tetramethylbenzidine may be

0 used; and in cases where alkaline phosphatase is used as an enzyme, para-nitrophenol or the like may be used. As a radioisotope, those used in an ordinary radioimmunoassay such as ¹²⁵I and ³H may be used. As a fluorescent dye, one used in an ordinary fluorescent antibody technique such as fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) or the like may be used.

[0068]

These immunoassays per se are well-known in the art, and so it is not necessary to explain these immunoassays in the present specification. Briefly, in

2017239516 04 Oct 2017 sandwich immunoassays, for example, the above-mentioned polypeptide used as an antigen is immobilized on a solid phase, and then reacted with a sample such as a serum. After washing the solid phase, the resultant is reacted with an appropriate secondary antibody. After washing the solid phase, the secondary antibody bound to the solid phase is measured. In the method for detecting a cancer(s) according to the present invention, it is preferred to immobilize an antigen polypeptide on a solid phase, because immobilization on a solid phase makes it possible to easily remove the unbound secondary antibody. As the secondary antibody, for example, anti-dog IgG antibody may be used in cases where the sample is obtained from dogs. The secondary antibody bound to a solid phase may be measured by labeling the secondary antibody with a labeling substance exemplified above. The thus measured amount of the secondary antibody corresponds to the amount of the abovementioned antibody in a serum sample. In cases where an enzyme is used as a labeling substance, the amount of the antibody may be measured by adding a substrate which is decomposed by the enzymatic activity to develop a color, and then optically measuring the amount of decomposed substrate. In cases where a radioisotope is used as a labeling substance, the amount of radiation from the radioisotope may be measured with a scintillation counter or the like.

[0069]

In Method 2 of the present invention, at least one polypeptide selected from the group consisting of the polypeptide of SEQ ID NO:2 or a homologous factor thereof, calmegin, CEP of SEQ ID NO:26 or 42 or a homologous factor thereof and TRIP11, which may be contained in a sample obtained from a living body, is measured. As explained above, the abundance of the cancer-specific antibody

5 which immunologically reacts with the polypeptide of SEQ ID NO :2 or a homologous factor thereof, calmegin in dogs, humans or the like, CEP of SEQ ID NO:26 or 42 or a homologous factor thereof, or TRIP11 in dogs, humans or the like

2017239516 04 Oct 2017 is significantly high in cancer patients, which indicates that the production of these polypeptides or homologous factors thereof, which are the antigen of the cancerspecific antibody, is significantly high in cancer patients. As concretely described in the Examples below, cancers can also be detected by measuring the antigen per se.

Therefore, similarly to Method 1 above, cancers in a living body can be detected by measuring the polypeptide of SEQ ID NO:2 or a homologous factor thereof, calmegin, CEP of SEQ ID NO:26 of 42 or a homologous factor thereof, or TRIP11 per se.

[0070]

Measurement of the polypeptide in a sample may easily be carried out by a well-known immunoassay. Specifically, for example, the polypeptide of SEQ ID NO :2,16,26,42 or 45 or a homologous factor thereof which may exist in a sample may be measured by preparing an antibody or antigen-binding fragment thereof which immunologically reacts with the polypeptide of SEQ ID NO:2,16, 26,42 or

45 or a homologous factor thereof, and then carrying out an immunoassay using the prepared antibody or fragment thereof. Because antibodies have a cross-reactivity as explained above, not only a canine-derived polypeptide of SEQ ID NO:2,16, 26, 42 or 45 but also a homologous factor in other mammals, for example, a human homologous factor of SEQ ID NO:4, 18,28 or 47 or a feline homologous factor, may be measured by using the antibody or antigen-binding fragment thereof which immunologically reacts with the canine-derived polypeptide of SEQ ID NO:2, 16,26, 42 or 45. Immunoassays per se are a well-known, conventional method as described above.

[0071]

5 The term antigen-binding fragment herein means fragment such as Fab fragment or F(ab')2 fragment of the antibody, which exhibits antigen-binding property of the antibody. Although the antibody may be either a polyclonal antibody or

2017239516 04 Oct 2017 monoclonal antibody, a monoclonal antibody is preferred for immunoassays and the like, because the reproducibility is high. Methods for preparing a polyclonal or monoclonal antibody using a polypeptide as an immunogen are well-known, and may be easily carried out by a conventional method. For example, antibodies against the 5 polypeptide may be induced by immunizing an animal with an immunogen, the polypeptide conjugated to a carrier protein such as keyhole limpet hemocyanin (KLH) or casein, together with an adjuvant. Then antibody-producing cells such as spleen cells or lymphocytes are collected from the immunized animal and fused with myeloma cells to prepare hybridomas. Among the hybridomas, one producing the 10 antibody which binds to the protein of SEQ ID NO:2, 16, 26,42 or 45 or a homologous factor thereof is selected and proliferated, and then the antibody whose corresponding antigen is the above-mentioned protein may be collected from the culture supernatant. The above-described method is a conventional well-known method.

[0072]

In Method 3 of the present invention, mRNA encoding any one of the polypeptides selected from the group consisting of the polypeptide of SEQ ID NO:2 or a homologous factor thereof, calmegin, CEP of SEQ ID NO:26 or 42 or a homologous factor thereof and TRIP11, which may be contained in a sample

0 obtained from a living body, is measured. As concretely described in the Examples below, the expression level of mRNA encoding the canine-derived polypeptide of SEQ ID NO:2 or the human homologous factor thereof shown in SEQ ID NO:4; mRNA encoding canine calmegin of SEQ ID NO: 16 or human calmegin of SEQ ID NO: 18; mRNA encoding canine CEP of SEQ ID NO:26 or 42 or the human

5 homologous factor thereof shown in SEQ ID NO:28; and mRNA encoding canine

TRIP11 of SEQ ID NO:45 or human TRIP11 of SEQ ID NO:47 is significantly high in cancer cells. Therefore, cancers in a living body can be detected by measuring

2017239516 04 Oct 2017 the mRNA in a sample.

[0073]

For example, mRNA in a sample may be quantified by a conventional method such as real-time detection RT-PCR using the mRNA as a template, and may also be roughly quantified based on the staining intensity in a conventional Northern blotting.

The sequence of cDNAs encoding the polypeptides of SEQ ID NOs:2,4, 16,18,26, 28,42,45 and 47 are shown in SEQ ID NOs:l, 3, 15, 17,25,27,41,44 and 46, respectively. Referring to these sequences, a polynucleotide which specifically hybridizes with a partial region of the base sequence shown in SEQ ID NO:1, 3,15, 10 17,25,27,41,44 or 46 (hereinafter referred to as polynucleotide for cancer detection) may be prepared, and using the polynucleotide as a probe or a primer for nucleic acid amplification, the amount of the mRNA in a sample may be measured. As explained below, mRNA encoding homologous factors in mammals other than dogs and humans may also be measured by using a polynucleotide which specifically 15 hybridizes with a partial region of the base sequence shown in SEQ ID NO: 1 or 3.

Similarly, mRNA encoding calmegin in mammals other than dogs and humans may also be measured by using a polynucleotide which specifically hybridizes with a partial region of the base sequence shown in SEQ ID NOHS or 17; mRNA encoding homologous factors in mammals other than dogs and humans may also be measured 20 by using a polynucleotide which specifically hybridizes with a partial region of the base sequence shown in SEQ ID NO :25,27 or 41; and mRNA encoding TRIP 11 in mammals other than dogs and humans may also be measured by using a polynucleotide which specifically hybridizes with a partial region of the base sequence shown in SEQ ID NO:44 or 46. In the present invention, polynucleotide 2 5 may be RNA or DNA.

[0074]

The term specifically hybridize used herein means that a certain sequence

2017239516 04 Oct 2017 hybridizes only with the subject partial region and does not substantially hybridize with the other regions under ordinary hybridization conditions.

[0075]

The term ordinary hybridization condition refers to a condition used for annealing in the ordinary PCR or the ordinary detection with probes. For example, in the case of PCR with Taq polymerase, the term refers to a reaction condition at an appropriate annealing temperature of about 54°C to 60°C using a common buffer such as one containing 50 mM KC1,10 mM Tris-HCl (pH 8.3 to 9.0) and 1.5 mM MgClz. In the case of Northern hybridization, the term refers to a reaction condition at an appropriate hybridization temperature of 42°C to 65°C using a common hybridization solution such as one containing 5 x SSPE, 50% formamide, 5 x Dehhardf s solution and 0.1 to 0.5% SDS. It should be noted, however, that the appropriate annealing temperature and hybridization temperature are not restricted to those exemplified above, and may be determined based on Tm of the primer or the probe and on the empirical rules. Those skilled in the art can easily determine the appropriate temperature.

[0076]

The term does not substantially hybridize means that a hybridization does not occur at all or, even if it occurs, the degree of the hybridization with regions other 2 0 than the subject partial region is considerably lower than that of the hybridization with the subject region so that the hybridization with other regions can be relatively ignored. Examples of the polynucleotide which specifically hybridizes under such conditions include those having a certain homology to the subject partial region, for example, those having a homology of not less than 70%, preferably not less than 80%, 2 5 more preferably not less than 90%, still more preferably not less than 93%, still more preferably not less than 95%, still more preferably not less than 98% to the subject partial region. Most preferably, the polynucleotide has the same base sequence as

2017239516 04 Oct 2017 the subject partial region. The same definition as for the homology of amino acid sequences applies to the homology of base sequences. Even if a polynucleotide for cancer detection comprises any region which does not hybridizes with the subject region at its end, a probe consisting of such a polynucleotide may be used for detecting cancers as long as a region which hybridizes with the subject region occupies about half or more of the whole probe. Similarly, a primer consisting of such a polynucleotide can normally anneal to the subject region to allow the extension reaction to occur and thus may be used for detecting cancers, as long as a region which hybridizes with the subject region occupies about half or more of the whole primer and is located at the 3'-end of the primer. It should be noted that, in cases where polynucleotides for cancer detection comprise any region which does not hybridizes with the subject region at its end, the homology to the subject base sequence is calculated based only on the region which hybridizes with the subject region, ignoring the non-hybridizing region.

[0077]

In the present invention, the term partial region refers to a region consisting of a part of the base sequence shown in SEQ ID NO: 1, 3,15, 17, 25, 27,41,44 or 46. A partial region preferably consists of not less than 18 consecutive bases. It is understood that base sequence shown in SEQ ID NO:1 as used herein includes not 2 0 only the base sequence expressly written in the SEQ ID NO: 1, but also the sequence complementary thereto. Thus, for example, the phrase a polynucleotide having the base sequence shown in SEQ ID NO: 1 includes a single strand polynucleotide having the base sequence expressly written in the SEQ ID NO :1, a single strand polynucleotide having the sequence complementary thereto, and a double strand

5 polynucleotide composed of these single strand polynucleotides. When the polynucleotides used in the present invention or polynucleotides encoding the polypeptides used in the present invention are prepared, any one of these base

2017239516 04 Oct 2017 sequences should be appropriately selected, and those skilled in the art can easily carry out the selection.

[0078]

In view of assuring specificity, the number of bases of the polynucleotide for cancer detection is preferably not less than 18 bases. In cases where the polynucleotide is used as a probe, the size is preferably not less than 18 bases, more preferably not less than 20 bases, and not more than the full length of the coding region. In cases where the polynucleotide is used as a primer, the size Is preferably not less than 18 bases, and preferably not more than 50 bases. Preferred examples of the polynucleotide for cancer detection include those consisting of not less than 18 consecutive bases of the base sequence shown in SEQ ID NO:1, 3,15, 17, 25, 27,41, 44 or 46.

[0079]

It is apparent for those skilled in the art who refer to the present specification that a polynucleotide which specifically hybridizes with a partial region of SEQ ID NO:1,15,25 or 44 is used for measurement of mRNA encoding a canine polypeptide of SEQ ID NO:2,16, 26 or 45, respectively; and that a polynucleotide which specifically hybridizes with a partial region of SEQ ID NO:3, 17,27 or 46 is used for a measurement of mRNA encoding a human homologous factor of SEQ ID NO;4, 18,

28 or 47, respectively. It should be noted that homologous factors usually share high homology with each other even at a base sequence level. For example, SEQ ID NOs:l and 3 share 93% homology, SEQ ID NOs:15 and 17 share 90% homology, SEQ ID NOs:25 and 27 share 87% homology, and SEQ ID NOs:44 and 46 share 88% homology, which are very high homology. Thus, a polynucleotide specifically

5 hybridizing with a partial region of SEQ ID NO: 1, 15, 25 or 44 may also specifically hybridize with the corresponding partial region of SEQ ID NO:3,17, 27 or 46, respectively. As practically demonstrated in the Examples below, for example, by

2017239516 04 Oct 2017 using a set of primers having the base sequences shown in SEQ ID NOs:7 and 8, respectively, mRNA encoding the canine-derived polypeptide of SEQ ID NO:2 and mRNA encoding the human homologous factor of SEQ ID NO :4 both may be measured, because the respective primers specifically hybridize with not only a partial region of SEQ ID NO: 1 but also a partial region of SEQ ID NO:3 (Example A). By using a set of primers having the base sequences shown in SEQ ID NOs: 19 and 20, respectively, mRNA encoding the canine calmegin of SEQ ID NO: 16 and mRNA encoding the human homologous factor, the human calmegin of SEQ ID NO: 18, both may be measured, because the respective primers specifically hybridize with not only a partial region of SEQ ID NO: 15 but also a partial region of SEQ ID NO: 17 (Example B). By using a set of primers having the base sequences shown in SEQ ID NOs:29 and 30, respectively, mRNA encoding the canine CEP of SEQ ID NO :26 or 42 and mRNA encoding the human homologous factor, the human CEP of SEQ ID NO:28, both may be measured, because the respective primers specifically hybridize with a partial region of SEQ ID NO:25, a partial region of SEQ ID NO:27, and also a partial region of SEQ ID NO:41 (Example C). By using a set of primers having the base sequences shown in SEQ ID NOs:48 and 49, respectively, mRNA encoding the canine TRIP 11 of SEQ ID NO:45 and mRNA encoding the human homologous factor, the human TRIP11 of SEQ ID NO :47, both may be measured,

0 because the respective primers specifically hybridize with not only a partial region of SEQ ID NO:44 but also a partial region of SEQ ID NO:46 (Example D). Thus, for example, by using the polynucleotide specifically hybridizing with a partial region of the canine base sequence shown in SEQ ID NO: 1; SEQ ID NO: 15; SEQ ID NO:25; or SEQ ID NO:44, not only mRNA encoding the canine polypeptide of SEQ ID

NO:2; SEQ ID NO: 16; SEQ ID NOs:26 and 42; or SEQ ID NO:45 but also mRNA encoding the human homologous factor thereof, the polypeptide of SEQ ID NO:4; SEQ ID NO: 18; SEQ ID NO:28; or SEQ ID NO:47, may be measured, respectively.

2017239516 04 Oct 2017

Furthermore, mRNA encoding the homologous factor in other mammals such as cats may also be measured by using the same polynucleotides.

[0080]

In designing a polynucleotide for cancer detection, it is more desirable to select a partial region in which homology between SEQ ID NOs: 1 and 3; SEQ ID NOs: 15 and 17; SEQ ID NOs:25 and 27; or SEQ ID NOs:44 and 46 is especially high (preferably a partial region having the same sequence). It is expected that a region especially highly homologous between dog and human also shares very high homology with a certain partial region of the homologous genes in other animal species. Therefore, by selecting a partial region in such a manner, the accuracy of measurement of mRNA which encodes homologous factors in animal species other than dog and human can be more improved.

[0081]

The methods per se for measuring a test nucleic acid using a polynucleotide which specifically hybridizes with a partial region of the test nucleic acid as a primer for a gene-amplification method such as PCR or as a probe are well-known, and include Northern blotting, in situ hybridization and the like, as well as RT-PCR as described in detail in the following Examples. Any of these well-known measurement methods may be used for measuring mRNA level in the present

0 invention.

[0082]

The nucleic acid-amplification methods per se such as PCR are well-known in the art, and the reagent kits and apparatuses are also commercially available, so that they may be easily carried out. That is, for example, a test nucleic acid serving 25 as a template (e.g., cDNA of the gene encoding the protein having the amino acid sequence shown in SEQ ID NO:2,4, 16,18,26, 28,45 or 47) and a pair of polynucleotides for cancer detection (primers) are mixed in a known buffer in the

2017239516 04 Oct 2017 presence of Taq polymerase and dNTP, and the steps of denaturation, annealing and extension are carried out by changing the temperature of the reaction mixture. Usually, the denaturation step is carried out at 90 to 95°C, the annealing step is carried out at Tm between the template and the primers or a vicinity thereof (preferably within ±4°C), and the extension step is carried out at 72°C which is the optimum temperature of Taq polymerase. The reaction time of each step is selected from about 30 seconds to 2 minutes. By repeating this thermal cycle for about 25 to 40 times, the region between a pair of the primers is amplified. The nucleic acidamplification method is not restricted to PCR, and other nucleic acid-amplification methods well-known in the art may also be employed. By carrying out the nucleic acid-amplification method using a pair of the above-described polynucleotides for cancer detection as primers and using the test nucleic acid as a template, the test nucleic acid is amplified. In contrast, in cases where the test nucleic acid is not contained in the sample, the amplification does not occur. Therefore, by detecting the amplification product, whether the test nucleic acid exists in the sample or not may be determined. Detection of the amplification product may be carried out by a method in which the reaction solution after the amplification is subjected to electrophoresis and then the bands are stained with ethidium bromide or the like, or by a method in which the amplification product after electrophoresis is immobilized on a solid phase such as a nylon membrane; a labeled probe which specifically hybridizes with the test nucleic acid is hybridized with the test nucleic acid; and then the label is detected after washing. Alternatively, the test nucleic acid in the sample may be quantified by the so-called realtime detection PCR using a quencher fluorescent dye and a reporter fluorescent dye. ' Since the kits for realtime detection

5 PCR are also commercially available, realtime detection PCR may also be carried out easily. The test nucleic acid may also be semi-quantified based on the intensity of the electrophoretic band. The test nucleic acid may be mRNA or cDNA reverse47

2017239516 04 Oct 2017 transcribed from mRNA. In cases where mRNA is amplified as the test nucleic acid, NASBA method (3 SR method, TMA method) using the above-described pair of primers may also be employed. NASBA method per se is well-known, and kits therefor are commercially available, so that NASBA method may easily be carried out using the above-described pair of primers.

[0083]

As the probe, a labeled probe obtained by labeling the above-described polynucleotide for cancer detection with a fluorescent label, radioactive label, biotin label or the like may be used. The methods per se for labeling a polynucleotide are 10 well-known. Whether the test nucleic acid exists in the sample or not may be determined by immobilizing the test nucleic acid or amplification product thereof on a solid phase, hybridizing the labeled probe therewith, and measuring the label bound to the solid phase after washing. Alternatively, the polynucleotide for cancer detection may be immobilized on a solid phase to hybridize the test nucleic acid therewith and detect the test nucleic acid bound to the solid phase by a labeled probe or the like. In such a case, the polynucleotide for cancer detection immobilized on the solid phase is also called a probe. The methods for measuring a test nucleic acid using a polynucleotide probe are also well-known in the art, and may be attained by bringing a polynucleotide probe into contact with the test nucleic acid in a buffer at

Tm or a vicinity thereof (preferably within ±4°C) so as to hybridize them, and then measuring the hybridized labeled probe or the test nucleic acid bound to the immobilized probe. Such a method includes well-known methods such as Northern blot and in situ hybridization, and Southern blot. Any of such known methods may be used in the present invention.

[0084]

In the detection method of the present invention, whether the subject living body suffers from cancer or not or the like is determined based on the expression

2017239516 04 Oct 2017 level of the polypeptide measured as described above. Although the cancer detection may be attained simply by measuring the expression of the polypeptide in the subject living body, it is preferred to obtain the normal reference value by determining the expression level of the polypeptide (the amount of the antibody, polypeptide or mRNA) in one or more samples from healthy individuals to compare the measured value in the subject living body with the normal reference value, in view of increasing the measurement accuracy. In order to further increase the measurement accuracy, the cancer reference value may be obtained by determining the expression level of the polypeptide in samples obtained from many patients who have been revealed to suffer from cancer to compare the measured value of the subject living body with both of the normal and cancer reference values. The above mentioned reference values may be determined by expressing the expression level of the polypeptide in each sample in values and calculating the average value thereof. The normal and cancer reference values may be determined beforehand by measuring the expression level of the polypeptide in many healthy and cancer subjects. Thus, the predetermined reference values may also be used when comparing the measured value with the reference values in the present invention.

[0085]

In cases where cancer detection is carried out based on the expression levels of two or more of the above-described four polypeptides, the subject living body may be judged to suffer from cancer when the expression level of any one polypeptide indicates cancer (see, Example E below).

[0086]

The detection method of the present invention may be carried out in

5 combination with diagnosis using other cancer antigens and/or cancer markers so that the detection accuracy of cancers can be more improved. For example, in measuring the above-mentioned cancer-specific antibody according to the present

2017239516 04 Oct 2017 invention, other polypeptide(s) highly expressed in cancer tissues may be used as an antigen in the same manner as the above-described polypeptides. The method of the present invention may also be carried out in combination with diagnosis using known cancer markers.

[0087]

By the detection method of the present invention, cancers in a living body can be detected. Especially, as described in the following Examples, the method of the present invention can detect even an invisible small cancer or a cancer which exists in a deep part of a body, and thus the method is useful for early detection of cancers.

Further, by applying the detection method of the present invention to patients in the follow-up period after cancer therapy, the recurrent cancer, if any, can be detect in its early stage, [0088]

If the more cancer cells expressing the prescribed polypeptide to be measured in the present invention proliferate in a cancer-bearing living body, the more the polypeptides and mRNAs encoding them accumulate in the body, which causes the increased amount of the antibodies against the above-mentioned polypeptides in the serum. On the other hand, the more cancer cells decrease, the more the accumulated polypeptides and mRNAs encoding them decrease in the body, which

0 causes the decreased amount of the antibodies against the above-mentioned polypeptides in the serum. Thus, if the expression level of the prescribed polypeptide is high, it can be determined that tumor growth and/or metastasis of cancer occurs, i.e., the stage of progression of cancer is advanced. Indeed, as concretely described in the Examples below, it was observed that the amount of the

5 above-mentioned antibody increases in the serum of cancer-bearing body along with the cancer progression such as tumor growth or metastasis. Hence, the stage of cancer progression can be detected by the method of the present invention.

2017239516 04 Oct 2017 [0089]

Further, as shown in the Example below, when compared between the same kind of tumors, a malignant one produces significantly more amount of the antibodies than a benign one. Therefore, if the expression level of the prescribed polypeptides is high, it can be determined that the grade of cancer malignancy is higher. That is, the grade of cancer malignancy can also be detected by the method of the present invention.

[0090]

Furthermore, the effect of the cancer therapy can be monitored based on the increase or decrease of the expression level of the prescribed polypeptides. As described in the Example below, compared to the cancer-bearing state, individuals receiving an anticancer drug for prevention of recurrence after tumor extirpation show decreased expression of the polypeptides. This applies to benign tumors. That is, in cases where the expression of the polypeptides can be observed, the decreased expression of the polypeptides is observed when complete extirpation of the benign tumor is attained. Therefore, by observing the expression level of the above-mentioned polypeptides on individuals during or after cancer therapy, a clue to assess how much the administered anticancer drug was effective, or whether a portion of the tumor is left in the patient after extirpation of the tumor can be

0 obtained, as well as a clue to find metastasis and/or recurrence as early as possible can be obtained during the follow-up. If cancer is appropriately treated in a patient, the expression level of the polypeptides becomes lower in the patient after therapy than before therapy. In such a case, it can be judged that the effect of the therapy which was (is being) performed on the patient is good. In cases where the

5 expression level of the polypeptides increases or is sustained, or once decreases and then increases, it can be judged that the effect of the therapy is not good enough. Thus, a useful basis for adoption of the therapeutic approach can be obtained. For

2017239516 04 Oct 2017 example, it can be judged, based on the above-described change of the expression level, whether the therapeutic approach should be changed to another, whether or how the dose of the anticancer drug should be changed, and so on.

[0091]

Cancers to be detected by the method of the present invention are those expressing at least one polypeptide selected from the group consisting of the polypeptide of SEQ ID NO:2 or a homologous factor thereof, calmegin, CEP of SEQ ID NO :26 or 42 or a homologous factor thereof, and TRIP11. Examples of the cancer to be detected include, but not limited to, brain tumor; squamous cell carcinomas of head, neck, lung, uterus and esophagus; melanoma; adenocarcinomas of lung and uterus; renal cancer; malignant mixed tumor; hepatocellular carcinoma; basal cell carcinoma; acanthomatous epulis; intraoral tumor; perianal adenocarcinoma; anal sac tumor; anal sac apocrine carcinoma; Sertoli cell tumor; vulva cancer; sebaceous adenocarcinoma; sebaceous epithelioma; sebaceous adenoma; sweat gland carcinoma; intranasal adenocarcinoma; nasal adenocarcinoma; thyroid cancer; colon cancer; bronchial adenocarcinoma; adenocarcinoma; ductal carcinoma; mammary adenocarcinoma; combined mammary adenocarcinoma; mammary gland malignant mixed tumor; intraductal papillary adenocarcinoma; fibrosarcoma; hemangiopericytoma; osteosarcoma; chondrosarcoma; soft tissue

0 sarcoma; histiocytic sarcoma; myxosarcoma; undifferentiated sarcoma; lung cancer; mastocytoma; cutaneous leiomyoma; intra-abdominal leiomyoma; leiomyoma; chronic lymphocytic leukemia; lymphoma; gastrointestinal lymphoma; digestive organ lymphoma; small cell or medium cell lymphoma; adrenomedullary tumor; granulosa cell tumor; pheochromocytoma; bladder cancer (transitional cell

5 carcinoma); suppurative inflammation; intra-abdominal liver tumor; liver cancer; plasmacytoma; malignant hemangiopericytoma; angiosarcoma; anal sac adenocarcinoma; oral cancer; metastatic malignant melanoma; amelanotic malignant

2017239516 04 Oct 2017 melanoma; cutaneous malignant melanoma; malignant myoepithelioma; malignant seminoma; seminoma; adenocarcinoma of the large intestine; gastric adenocarcinoma; low-grade sebaceous carcinoma; ceruminous adenocarcinoma; apocrine carcinoma; poorly differentiated apocrine sweat gland carcinoma; malignant fibrous histiocytoma; multiple myeloma; mesenchymal malignant tumor; liposarcoma; osteosarcoma; sarcoma of unknown origin; soft part sarcoma (spindle cell tumor); poorly differentiated sarcoma; synovial sarcoma; angiosarcoma; metastatic malignant epithelioma; tubular mammary adenocarcinoma; mammary ductal carcinoma; inflammatory breast cancer; germinoma; leukemia; invasive trichoepithelioma; medium cell lymphoma; multicentric lymphoma; osteosarcoma (mammary gland); mastocytoma (Patnaik II type); mastocytoma (Grade II); and leiomyosarcoma. The living bodies to which the method of the present invention applies are mammals, preferably humans, dogs and cats.

[0092]

The sample subjected to the method of the present invention include body fluids such as blood, serum, plasma, ascites and pleural effusion, and tissues and cells. Particularly, serum, plasma, ascites and pleural effusion may be preferably used in Method 1 and Method 2 above. A tissue sample and cell sample are preferred in the case of Method 3 above in which mRNA is measured.

[0093]

The polypeptides used as an antigen for immunoassay in Method 1 (i.e., a canine-derived polypeptide of SEQ ID NO:2, SEQ ID NO: 16, SEQ ID NO:26 or 42 or SEQ ID NO :4 5 and homologous factors thereof, specifical ly-reactive partial polypeptides, specifically-reactive modified polypeptides, and specifically-reactive

5 added polypeptides) may be provided as a reagent for detecting a cancer(s). The reagent may consist only of the above-mentioned polypeptide, or may contain various additives useful for stabilizing the polypeptide and the like. The reagent may also

2017239516 04 Oct 2017 be provided in the form of being immobilized on a solid phase such as a plate or membrane.

[0094]

The antibodies or antigen-binding fragments thereof which immunologically react with the canine polypeptide of SEQ ID NO:2, SEQ ID NO: 16, SEQ ID NO:26 or 42 or SEQ ID NO:45 or a homologous factor thereof, which are used for measuring the canine polypeptide or the homologous factor thereof by immunoassay, may also be provided as a reagent for detecting a cancer(s). The reagent may also consist only of the above-mentioned antibody or antigen-binding fragment thereof, or 10 may contain various additives useful for stabilizing the antibody or antigen-binding fragment thereof and the like. The antibody or antigen-binding fragment thereof may also be in the form of being conjugated with a metal such as manganese or iron. Since such a metal-conjugated antibody or antigen-binding fragment thereof accumulates in a site in which a large amount of antigen protein exists when administered to a body, the existence of cancer cells which produce the antigen protein can be detected by measuring the metal by MRI or the like.

[0095]

Furthermore, the above-described polynucleotides for cancer detection used for measuring mRNA in Method 3 may also be provided as a reagent for detecting a 2 0 cancer(s). The reagent may also consist only of the polynucleotide, or may contain various additives useful for stabilizing the polynucleotide and the like. The polynucleotide for cancer detection contained in the reagent is preferably a primer or a probe. The conditions and preferred examples of the polynucleotide for cancer detection are as already described above.

EXAMPLES [0096]

The present invention will now be described more concretely by way of

2017239516 04 Oct 2017

Examples.

[0097]

Example A-l: Acquisition of Novel Cancer Antigen Protein by SEREX Method (1) Preparation of cDNA Library

Total RNA was prepared from testis tissue of a healthy dog by the Acid guanidium-Phenol-Chloroform method, and poly(A) RNA was purified using Oligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co., Ltd.) in accordance with the protocol attached to the kit.

[0098]

Using the obtained mRNA (5 pg), a dog testis cDNA phage library was synthesized. Preparation of the cDNA phage library was carried out using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit (manufactured by STRATAGENE) in accordance with the protocols attached to the kits. The size of the prepared cDNA phage library was 1.3 χ 10⁶ pfu/ml.

[0099] (2) Screening of cDNA Library with Serum

Using the dog testis-derived cDNA phage library prepared as described above, immunoscreening was carried out. More particularly, host E. coli cells (XL 1-Blue MRF') were infected with the library such that 2,340 clones should appear on an

0 NZY agarose plate having the size of Φ90 * 15 mm, and cultured at 42°C for 3 to 4 hours to allow the phage to form plaques. The plate was covered with nitrocellulose membrane (Hybond C Extra: manufactured by GE Healthcare Bio-Science) impregnated with IPTG (isopropyl-β-D-thiogalactoside) at 37°C for 4 hours to induce and express proteins, which were thus transferred to the membrane. Subsequently,

5 the membrane was recovered and soaked in TBS (10 mM Tris-HCl, 150 mM NaCl; pH 7.5) containing 0.5% non-fat dry milk, followed by shaking it at 4°C overnight to suppress non-specific reactions. This filter was allowed to react with 500-fold

2017239516 04 Oct 2017 diluted canine patient serum at room temperature for 2 to 3 hours.

[0100]

As the above-described canine patient serum, serum collected from canine patients suffering from squamous cell carcinoma was used. The serum was stored at -80°C and pretreated immediately before use. The method of the pretreatment of the serum was as follows. That is, host E. coli cells (XL1 -Blue MRF') were infected with λ ZAP Express phage to which no foreign gene was inserted, and then cultured on NZY plate medium at 37°C overnight. Subsequently, the buffer of 0.2 M NaHCC>3, pH 8.3 containing 0.5 M NaCl was added to the plate, and the plate was left to stand at 4°C for 15 hours, followed by collecting the supernatant as an E. coZz'/phage extract. Thereafter, the collected E. coZz/phage extract was allowed to flow through an NHS column (manufactured by GE Healthcare Bio-Science) to immobilize proteins derived from the E. coZz/phage thereon. The serum from the canine patients was allowed to flow through and react with this protein-immobilized column to remove antibodies adsorbed on E. coli and/or the phage. The serum fraction that passed through the column was 500-fold diluted with TBS containing 0.5% non-fat dry milk, and the resulting diluent was used as the material for the immunoscreening.

[0101]

0 The membrane on which the thus treated serum and the above-described fusion protein were blotted was washed 4 times with TBS-T (0.05% Tween 20/TBS), and allowed to react with goat anti-dog IgG (Goat anti Dog IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5,000-fold diluted with TBS containing 0.5% non-fat dry milk as a secondary antibody at room temperature for 1 hour,

5 followed by detection by the enzyme coloring reaction using the NBT/BCIP reaction solution (manufactured by Roche). Colonies at positions where a positive coloring reaction was observed were recovered from the NZY agarose plate having the size of

2017239516 04 Oct 2017

Φ90 χ 15 mm, and dissolved in 500 μΐ of SM buffer (100 mM NaCl, 10 mM MgClSCfi, 50 mM Tris-IICl, 0.01% gelatin; pH 7.5). The screening was repeated as a second and third screening in the same manner as described above until a single coloring reaction-positive colony was obtained, thereby isolating one positive clone after screening of 30,940 phage clones reactive with IgG in the serum.

[0102] (3) Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-described method to a base sequence analysis, an operation of conversion of the phage vector to a plasmid 10 vector was carried out. More particularly, 200 μΐ of a solution prepared to contain a host E. coli (XL 1-Blue MRF') such that the absorbance ODgoo should be 1.0 was mixed with 100 μΐ of a purified phage solution and further with 1 μΐ of ExAssist helper phage (manufactured by STRATAGENE), and the reaction was allowed to proceed at 37°C for 15 minutes. To the reaction mixture, 3 ml of LB medium was added, and the mixture was cultured at 37°C for 2,5 to 3 hours, followed by immediate incubation in a water bath at 70°C for 20 minutes. The mixture was then centrifuged at 4°C at 1,000 *g for 15 minutes, and the supernatant was recovered as a phagemid solution. Subsequently, 200 μΐ of a solution prepared to contain a phagemid host E. coli (SOLR) such that the absorbance ODgoo should be 1,0 was

0 mixed with 10 μΐ of a purified phage solution, and the reaction was allowed to proceed at 37°C for 15 minutes. Thereafter, 50 μΐ of the reaction mixture was plated on ampicillin (final concentration: 50 ug/m!)-contaming LB agar medium, and cultured at 37°C overnight. A single colony of transformed SOLR was recovered and cultured in ampicillin (final concentration: 50 pg/ml)-containing LB medium at

5 37°C, followed by purification of plasmid DNA having an insert of interest using

QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

[0103]

2017239516 04 Oct 2017

The purified plasmid was subjected to an analysis of the entire sequence of the insert by the primer walking method using the T3 primer described in SEQ ID NO:5 and the T7 primer described in SEQ ID NO:6. By this sequence analysis, the gene sequence described in SEQ ID NO: 1 was obtained. Using the base sequence and the amino acid sequence of this gene, homology search against known genes was carried out using a homology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, it was revealed that the obtained gene is the gene (Accession No. XM_535343) encoding a protein (Accession No.XP 535343) whose function is unknown. The human homologous factor of this gene was the gene (Accession No.NM_l 52660) encoding a protein (Accession No.NP_689873) whose function is also unknown (homology: base sequence, 93%; amino acid sequence, 99%). The base sequence of the human homologous factor is shown in SEQ ID NO:3, and the amino acid sequence thereof is shown in SEQ ID NO:4.

[0104] (4) Analysis of Expression in Each Tissue

The expression of the gene, which was obtained by the above-described method, in normal tissues and various cell lines of dog and human were investigated by the RT-PCR (Reverse Transcription-PCR) method. The reverse transcription 2 0 reaction was carried out as follows. That is, total RNA was extracted from 50 to 100 mg of each tissue or 5 to 10 * 10⁶ cells of each cell line using TRIZOL reagent (manufactured by Invitrogen) in accordance with the protocol attached to the kit. Using this total RNA, cDNA was synthesized by Superscript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) in accordance with the protocol 2 5 attached to the kit. As the cDNAs from human normal tissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA (manufactured by Invitrogen), QUICKClone cDNA (manufactured by CLONTECH) and Large-Insert cDNA Library

2017239516 04 Oct 2017 (manufactured by CLONTECH) were used. The PCR reactions were carried out as follows using primers (described in SEQ ID NOs:7 and 8) specific to the obtained canine gene and its human homologous gene. That is, respective reagents and the attached buffer were mixed such that the mixture should contain 0.25 μΐ of the sample prepared by the reverse transcription reaction, 2 μΜ each of the above primers, 0.2 mM each of dNTP and 0.65 U of ExTaq polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 25 μΐ, and the reaction was carried out with 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute using Thermal Cycler (manufactured by BIO RAD). The gene-specific primers having the base sequences shown in the above-described SEQ ID NOs:7 and 8 were those which amplify the regions of the 87th to 606th bases of the base sequence of SEQ ID NO:1 and the 173rd to 695th bases of the base sequence of SEQ ID NO:3, and can be used for investigation of the expression of both the canine gene and its human homologous gene. As a control for comparison, primers (described in SEQ

ID NOs:9 and 10) specific to GAPDH were used simultaneously. As a result, as shown in Fig. 1, strong expression of the obtained canine gene was observed in testis among the normal dog tissues, and on the other hand, strong expression was observed in the canine breast cancer cell line. Expression of the human homologous gene was confirmed, as is the case with the canine gene, only in testis among the human

0 normal tissues, but the expression was detected in brain tumor, leukemia, breast cancer and lung cancer cells among human cancer cell lines. Thus, the human homologous gene was also confirmed to be specifically expressed in testis and cancer cells.

[0105]

In Fig. 1, reference numeral 1 in the ordinate indicates the expression pattern of the above identified gene, and reference numeral 2 indicates the expression pattern of the GAPDH gene as a control for comparison.

2017239516 04 Oct 2017 [0106]

Example A-2: Preparation of Novel Cancer Antigen Proteins (1) Preparation of Recombinant Protein

Based on the gene of SEQ ID NO: 1 obtained in Example A-1, a recombinant protein was prepared by the following method. Respective reagents and the attached buffer were mixed such that the mixture should contain 1 μΐ of the vector which was prepared from the phagemid solution obtained in Example A-l and was subjected to the sequence analysis, 0.4 μΜ each of two kinds of primers having Ndel and Xhol restriction sites (described in SEQ ID NOs:l 1 and 12), 0.2 mM dNTP and

1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 50 μΐ, and PCR was carried out with 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds and 72°C for 1 minute using Thermal Cycler (manufactured by BIO RAD). Using the above-described two kinds of primers, the region encoding the entire amino acid sequence of SEQ ID NO:2 is obtained. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 930 bp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0107]

The purified DNA fragment was ligated into a cloning vector pCR-Blunt

0 (manufactured by Invitrogen). E. coli was transformed with the resulting ligation product, and plasmids were recovered thereafter, followed by confirming, by sequencing, that the amplified gene fragment matches the sequence of interest. The plasmid that matched the sequence of interest was treated with restriction enzymes Ndel and Xhol and purified using QIAquick Gel Extraction Kit, followed by inserting

5 the gene sequence of interest into an expression vector for E. coli, pET16b (manufactured by Novagen) that had been treated with Ndel and ATzoI. Usage of this vector enables production of a His-tag fusion recombinant protein. E. coli for

2017239516 04 Oct 2017 expression, BL21 (DE3), was transformed with this plasmid, and expression of the protein of interest was induced in E. coli with 1 mM IPTG.

[0108]

On the other hand, based on the gene of SEQ ID NO:3, a recombinant protein of the human homologous gene was prepared by the following method. Respective reagents and the attached buffer were mixed such that the mixture should contain 1 μΐ of the cDNA prepared in Example A-l whose expression could be confirmed by the RT-PCR method in various tissues/cells, 0.4 μΜ each of two kinds of primers having EcoRV and EcoRI restriction sites (described in SEQ ID NOs:13 and 14), 0.2 mM dNTP and 1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 50 pl, and PCR was carried out with 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds and 72°C for 1 minute using Thermal Cycler (manufactured by BIO RAD). Using the above-described two kinds of primers, the region encoding the entire amino acid sequence of SEQ ID NO:4 is obtained. After the PCR, the amplified DNA was subjected to electrophoresis using 1 % agarose gel, and a DNA fragment of about 930 bp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0109]

The purified DNA fragment was ligated into a cloning vector pCR-Blunt

0 (manufactured by Invitrogen). E. coli was transformed with the resulting ligation product, and plasmids were recovered thereafter, followed by confirming, by sequencing, that the amplified gene fragment matches the sequence of interest. The plasmid that matched the sequence of interest was treated with restriction enzymes L’coRV and KcoRI and purified using QIAquick Gel Extraction Kit, followed by

5 inserting the gene sequence of interest into an expression vector for E. coli, pET30a (manufactured by Novagen) that had been treated with £coRV and EcoRI. Usage of this vector enables production of a His-tag fusion recombinant protein. E. coli for

2017239516 04 Oct 2017 expression, BL21 (DE3), was transformed with this plasmid, and expression of the protein of interest was induced in E. coli with 1 mM IPTG.

[0110] (2) Purification of Recombinant Protein

The above-obtained recombinant E. coli cells that expressed SEQ ID NO: 1 and SEQ ID NOG, respectively, were cultured in ampicillin (final concentration: 100 pg/ml)-containing LB medium at 37°C until the absorbance at 600 nm reached about 0.7, and then IPTG was added thereto such that its final concentration should be 1 mM, followed by culturing them at 37°C for 4 hours. Subsequently, the cells were collected by centrifugation at 4,800 rpm for 10 minutes. The pellet of the cells was suspended in phosphate-buffered saline and further subjected to centrifugation at 4,800 rpm for 10 minutes to wash the cells.

[0111]

The cells were suspended in 50 mM Tris-HCl buffer (pH 8.0) and subjected to sonication on ice. The sonicated solution of E. coli was centrifuged at 6,000 rpm for 20 minutes to obtain the supernatant as the soluble fraction and the precipitate as the insoluble fraction.

[0112]

The insoluble fraction was suspended in 50 mM Tris-HCl buffer (pH 8.0) and

0 centrifuged at 6,000 rpm for 15 minutes. This operation was repeated twice and an operation of removal of proteases was carried out.

[0113]

The residue was suspended in 6M guanidine hydrochloride (manufactured by Sigma Aldrich Japan), 0.15 M sodium chloride-containing 50 mM Tris-HCl buffer

5 (pH 8.0), and the resulting suspension was left to stand at 4°C for 15 hours to denature proteins. Thereafter, the suspension was centrifuged at 6,000 rpm for 30 minutes, and the obtained soluble fraction was placed in a nickel chelate column

2017239516 04 Oct 2017 prepared by a conventional method (carrier: Chelating Sepharose (trademark) Fast Flow (GE Health Care); column volume: 5mL; equilibration buffer: 6M guanidine hydrochloride, 0.15 M sodium chloride-containing 50 mM Tris-HCl buffer (pH 8.0)), followed by leaving it to stand at 4°C overnight to allow adsorption to the nickel5 chelated carrier. The supernatant was recovered by centrifugation of this column carrier at 1,500 rpm for 5 minutes, and the column carrier was suspended in phosphate-buffered saline, followed by refilling the column with the resulting suspension.

[0114]

The fraction that was not adsorbed to the column was washed away with 10 column volumes of 0.5 M sodium chloride-containing 0.1 M acetate buffer (pH 4.0), and elution was immediately carried out with 0,5 M sodium chloride-containing 0.1 M acetate buffer (pH 3.0). Six column volumes of the eluted fraction was collected in each elution step. Elution of the proteins of interest was confirmed by Coomassie staining carried out according to a conventional method. Based on the result, the eluted fractions were desalted and concentrated to obtain the material to be solidphased for diagnosis.

[0115]

Example A-3: Cancer Diagnosis Using Recombinant Canine Protein (1) Cancer Diagnosis in Dogs

Blood samples were collected from 486 canine patients in which malignant or benign tumors were found and 6 healthy dogs, and sera were separated therefrom. Using the recombinant canine protein prepared in Example A-2 and anti-dog IgG antibody, the IgG antibody titer of the sera which specifically react with the

5 recombinant protein was measured by ELISA.

[0116]

As for immobilization of the prepared protein on a solid phase, 100 pL/well

2017239516 04 Oct 2017 of a solution of the recombinant protein diluted to 50 pg/mL with phosphate buffered saline was added to a 96-well Immobilizer Amino plate (manufactured by Nunc), and the plate was left to stand at 4°C overnight. As for blocking, 100 pL/well of 50 mM sodium bicarbonate buffer (pH 8.3) containing 0.5% BSA (bovine serum albumin, manufactured by Sigma Aldrich Japan) (hereinafter referred to as blocking solution) was added to the plate, and the plate was shaken at room temperature for 1 hour. Serum was 500-fold diluted with the blocking solution, and 100 uL/well of the diluted serum was added to the plate, followed by shaking the plate at room temperature for 3 hours to allow the reaction to proceed. After washing the wells 3 times with phosphate buffered saline containing 0.05% Tween20 (manufactured by Wako Pure Chemicals) (hereinafter referred to as PBS-T), 100 pL/well of HRPconjugated dog IgG antibody (Goat anti Dog IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 3,000-fold diluted with the blocking solution was added thereto, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed.. After washing the wells 3 times with PBS-T, 100 μΐ/well of a HRP substrate, TMB (1-Step Turbo TMB (tetramethylbenzidine), manufactured by PIERCE), was added thereto, and the enzyme-substrate reaction was allowed to proceed at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding 100 μΙ/well of 0.5 M sulfuric acid solution (manufactured by Sigma

0 Aldrich Japan), and then the absorbance was measured at 450 nm with a microplate reader. As a control, a plate on which the prepared recombinant protein was not immobilized and a plate with which the serum from a cancer-bearing dog was not reacted were measured in the same manner as above.

[0117]

5 Among the total 486 samples used in the above-described cancer diagnosis,

311 samples were definitely diagnosed as malignant by pathological diagnosis using the extirpated tumor tissue.

2017239516 04 Oct 2017 [0118]

Specifically, the samples were diagnosed as cancer such as malignant melanoma; malignant mixed tumor; hepatocellular carcinoma; basal cell carcinoma; acanthomatous epulis; intraoral tumor; perianal adenocarcinoma; anal sac tumor; anal 5 sac apocrine carcinoma; Sertoli cell tumor; vulva cancer; sebaceous adenocarcinoma;

sebaceous epithelioma; sebaceous adenoma; sweat gland carcinoma; intranasal adenocarcinoma; nasal adenocarcinoma; thyroid cancer; colon cancer; bronchial adenocarcinoma; adenocarcinoma; ductal carcinoma; mammary adenocarcinoma; combined mammary adenocarcinoma; mammary gland malignant mixed tumor;

intraductal papillary adenocarcinoma; fibrosarcoma; hemangiopericytoma; osteosarcoma; chondrosarcoma; soft tissue sarcoma; histiocytic sarcoma; myxosarcoma; undifferentiated sarcoma; lung cancer; mastocytoma; cutaneous leiomyoma; intra-abdominal leiomyoma; leiomyoma; squamous cell carcinoma; chronic lymphocytic leukemia; lymphoma; gastrointestinal lymphoma; digestive organ lymphoma; small cell or medium cell lymphoma; adrenomedullary tumor; granulosa cell tumor; pheochromocytoma; bladder cancer (transitional cell carcinoma); suppurative inflammation; intra-abdominal liver tumor; liver cancer; plasmacytoma; malignant hemangiopericytoma; angiosarcoma; anal sac adenocarcinoma; oral cancer; metastatic malignant melanoma; amelanotic malignant

0 melanoma; cutaneous malignant melanoma; malignant myoepithelioma; malignant seminoma; seminoma; adenocarcinoma of the large intestine; gastric adenocarcinoma; low-grade sebaceous carcinoma; ceruminous adenocarcinoma; apocrine carcinoma; poorly differentiated apocrine sweat gland carcinoma; malignant fibrous histiocytoma; multiple myeloma; mesenchymal malignant tumor;

5 iipo sarcoma; osteosarcoma; sarcoma of unknown origin; soft part sarcoma (spindle cell tumor); poorly differentiated sarcoma; synovial sarcoma; angiosarcoma; metastatic malignant epithelioma; tubular mammary adenocarcinoma; mammary

2017239516 04 Oct 2017 ductal carcinoma; inflammatory breast cancer; germinoma; leukemia; invasive trichoepithelioma; medium cell lymphoma; multicentric lymphoma; osteosarcoma (mammary gland); mastocytoma (Patnaik II type); mastocytoma (Grade II); leiomyosarcoma or the like.

[0119]

As shown in Fig. 3, sera from these cancer-bearing dogs showed a significantly high antibody titer against the recombinant protein. It was revealed that, by diagnosing a sample showing twice the average value of healthy canine samples as malignant, 192 samples i.e. 61.7% of the malignant cases could be successfully diagnosed as malignant. The details of these 192 cancer samples are as follows. It is noted that the following number of each cancer case is a cumulative total, as some samples contained multiple primaries.

Malignant melanoma, 10 cases; lymphoma, 9 cases; pheochromocytoma, 1 case; granulosa cell tumor, 1 case; hepatocellular carcinoma, 3 cases; angioma, 1 case;

malignant testicular tumor, 9 cases; intraoral tumor, 4 cases; perianal adenocarcinoma, 7 cases; osteosarcoma, 3 cases; fibrosarcoma, 8 cases; ductal carcinoma, 19 cases; chondrosarcoma, 1 case; mammary adenocarcinoma, 35 cases; combined mammary adenocarcinoma, 24 cases; lung cancer, 1 case; sebaceous adenocarcinoma, 2 cases; nasal adenocarcinoma, 2 cases; mastocytoma, 26 cases;

adrenomedullary tumor, 1 case; leiomyosarcoma, 2 cases; squamous cell carcinoma, cases; chronic lymphocytic leukemia, 1 case; undifferentiated sarcoma, 1 case; malignant mixed tumor, 2 cases; hemangiopericytoma, 1 case; tumor in the left knee joint, 1 case; tumor in the posterior segment of the left lobe of the lung, 1 case; bladder cancer (transitional cell carcinoma), 1 case; soft part sarcoma (spindle cell

5 tumor), 1 case; ceruminous adenocarcinoma, 1 case; multicentric lymphoma, 2 cases; liposarcoma, 1 case; synovial sarcoma, 1 case; invasive trichoepithelioma, 1 case; anal sac adenocarcinoma, 1 case.

2017239516 04 Oct 2017 [0120]

The above-described diagnostic method was also carried out using pleural effusion samples and ascites samples collected from terminal cancer dogs. As a result, values similar to those detected in serum samples could be detected, and hence 5 cancer diagnosis could be successfully attained.

[0121]

Furthermore, it was confirmed that diagnostic approaches such as diagnosis of cancers existing in an invisible part of the body, assessment of cancer stage and grade, follow-up of postoperative patients, diagnosis of recurrence and metastasis and the 10 like can also be attained by applying the above-described diagnostic method. The followings are several of the practical examples of the detailed diagnosis shown in Fig. 4.

[0122] (2)-1 Diagnosis of Invisible Tumors

In Canine Patient 1 (Flat-Coated Retriever), any tumors were not found on

June 7,2007. But about 20 days later, on June 24,2007, a pedunculated tumor with a diameter of 2 mm was found on the gum at the root of the canine tooth. The tumor was ligated at its pedunculated part and excised on the day it found. The absorbance at 450 nm observed before the tumor became visible with the naked eye

0 was 0.32, which was significantly high and not so different from the absorbance at the time of finding tumor, 0.37. The result indicates that it is possible to diagnose cancers even in an invisible part such as an intraperitoneal part by the method of the present invention.

[0123]

5 Rise of the value was observed before the tumor became visible with the naked eye, which is considered to have been a sign of tumor development. Thus, the method of the present invention is useful in medical examinations such as

2017239516 04 Oct 2017 periodic health examination.

[0124]

Canine Patient 1 was again checked by the serodiagnosis 2 weeks after the tumor excision. As a result, the absorbance at 450 nm greatly decreased to 0.07.

Thus, it was also confirmed that the cancer antigen-expressing tumor which had caused the increased antibody titer was completely removed (see, (2)-4, Follow-Up of Postoperative Patients).

[0125] (2)-2 Assessment of Stage of Cancer Progression

The stage of cancer progression is determined based on the size or depth of the tumor, how much the tumor exerts influence on the surrounding tissues, whether the tumor metastasizes or not, and the like. It was revealed herein that the detected value is higher than before if the metastasis occurs, i.e., the cancer has advanced. The following is another example of assessment of stage of a certain cancer case, which received anticancer drug therapy.

[0126]

Canine Patient 2 (Mixed Breed) underwent tumor extirpation by amputating the right hind leg on October 13,2006. According to the pathological diagnosis using the extirpated tumor, it was a highly-malignant mastocytoma at Grade II rather 2 0 close to Grade III. On March 12,2007, metastasis and recurrence were found in the right groin and liver, and anticancer drug therapy (vinblastine and prednisolone) was started without any surgical operations. Administration of anticancer drugs was started at the time of finding the metastasis and recurrence, and drugs were administered 1, 2,4 and 8 weeks thereafter again. The serodiagnosis was carried

5 out each time drugs were administered to find that the absorbance at 450 nm was

0.36,0.37, 0.26, 0.20 and 0.29, respectively. The value gradually decreased with anticancer drugs administered with short intervals from the start to the 4th week,

2017239516 04 Oct 2017 which indicates that the progression of cancer could be suppressed. However, the value increased again in the 8th week, when 1 month had passed since the previous administration, which indicates that the cancer began to advance again. It was also confirmed clinically that the tumor grew larger at that time. The result obtained in

Canine Patient 2 revealed that the stage of cancer progression can also be assessed by this method, and that the effect of anticancer drug therapy can also be assessed as shown above.

[0127] (2)-3 Assessment of Grade of Cancer Malignancy

Basaliomas include malignant type and benign type. Recently, according to the new WHO classification, malignant basaliomas are called basal cell carcinoma, and benign basaliomas are called trichoblastoma.

[0128]

Canine Patient 3 (Beagle) was diagnosed as basal cell carcinoma (malignant).

The serodiagnosis was carried out at the time of the surgery to find that the absorbance at 450 nm was 0.35. On the other hand, in Canine Patient 4 (Mixed Breed) diagnosed as trichoblastoma (benign), the serodiagnosis carried out at the time of the surgery revealed that the absorbance at 450 nm was 0, not detected at all. Thus, even in the case of the same basaliomas, malignant basal cell carcinoma and

0 benign trichoblastoma can be distinctively diagnosed.

[0129]

Next example is mammary gland tumors. Mammary gland tumors include malignant tumors such as mammary adenocarcinoma and mammary gland malignant mixed tumor, and benign mammary tumors which do not show malignant symptoms.

5 Canine Patient 5 (Yorkie) underwent extirpation of mammary gland malignant mixed tumor and mammary adenocarcinoma on May 17,2006. In general, the complete excision of mixed tumors in mammary gland is easy because they are poorly invasive

2017239516 04 Oct 2017 to the surrounding tissues even if they are malignant, and thus the postoperative course of the patients is usually uneventful. However, Canine Patient 5 had been diagnosed as highly malignant tumor, because the pathological diagnosis using the extirpated tissue revealed that some components of the specimen from Canine Patient 5 5 showed an invasive nature. On the other hand, mammary adenocarcinoma is a highly invasive tumor which often recurs and metastasizes. Although invasion of the tumor cells was not observed in the specimen from Canine Patient 5, it had been pointed out that highly malignant components possibly proliferated in other region out of the specimen. Thus, the findings in the pathological diagnosis clearly taught that Canine Patient 5 was suffering from highly malignant mammary cancer. A blood sample was collected during the surgery and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0.39. Canine Patient 6 (Yorkshire Terrier) underwent extirpation of mammary tumor on January 28,2007. According to the pathological diagnosis using the extirpated tissue, atypism of cells was low, and thus Canine Patient 6 was diagnoses as benign mastadenoma without malignant findings. A blood sample was collected during the surgery and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0.05. The results in the two cases above revealed that highly malignant tumors show a higher value than low malignant, benign tumors.

[0130] (2)-4 Follow-Up of Postoperative Patients

Canine Patient 7 (Shih Tzu) visited the hospital due to an intraoral tumor and underwent the extirpation on March 22,2007. The serodiagnosis was carried out at that time to find that the absorbance at 450 nm was 0.40. In addition, based on the

5 pathological diagnosis using the extirpated tissue, Canine Patient 7 was diagnosed as malignant acanthomatous epulis. This kind of tumor often recurs if excision is insufficient, though distant metastasis seldom happens. Thus, it is important

2017239516 04 Oct 2017 whether the tumor can be completely excised by surgery or not. According to the follow up on May 18,2007, the absorbance at 450 nm was 0.25, and hence the antibody titer was decreased. The recurrence has not been found till August of 2007. Thus, it is considered that the value obtained in the serodiagnosis became lower than that obtained at the time of surgery because the tumor could be completely excised from Canine Patient 7.

[OBI] (2)-5 Diagnosis of Recurrence

Canine Patient 8 (Husky) underwent an extirpation of mammary adenocarcinoma on May 8, 2007. The serodiagnosis was carried out at the time of the surgery to find that the absorbance at 450 nm was 0.08. The pathological diagnosis using the extirpated tissue revealed that highly atypical epithelial cells proliferated and mainly formed ductal structures, and thus this patient was diagnosed as primary breast adenocarcinoma. It was said that the patient was at a high risk of recurrence or metastasis to lymph nodes or distant organs, as many cancer cells had already entered the lymph vessels at that time. On June 28,2007, about 1-and-ahalf month after the surgery, metastasis was found at the same site. The value detected by the serodiagnosis carried out then was 0.08, which did not decrease at all. Thus, it is considered that the value of the serodiagnosis stayed unchanged from the

0 beginning of May to the end of June because the tumor could not have been completely excised or recurrence would have occurred in Canine Patient 8. [0132] (2)-6 Diagnosis of Metastasis

Canine Patient 9 (Scottish Terrier), repeatedly undergoing metastasis and

5 recurrence, was diagnosed as mammary tumor in February of 2003; intraoral malignant melanoma in August of 2003; malignant melanoma of the lip in January of 2005; and as intraoral melanoma on April 13,2005, all of which were excised by

2017239516 04 Oct 2017 surgery. This patient visited the hospital again on December 17,2006 for follow-up after the recurrence of intraoral melanoma in April of 2005, and the serodiagnosis was carried out at that time to find that the absorbance at 450 nm was 0.3. Half a year later, on June 20,2007, the patient again visited the hospital because of the hypertrophy of cervical and malar lymph nodes. In the case of lymphomas, hypertrophy of lymph nodes is systemically observed. Because Canine Patient 9 had only two swollen lymph nodes, this patient was clinically diagnosed as probable metastatic lymphoma. The diagnosis according to the present invention also revealed that it was a tumor which had metastasized from the tumor previously existed in this patient as the absorbance at 450 nm greatly increased to 0.75.

[0133] (2)-7 Therapy Monitoring

Canine Patient 11 (Miniature Dachshund) underwent tumor extirpation on

April 19,2007. According to the pathological diagnosis using the extirpated tumor, the patient was suffering from moderately-malignant combined mammary adenocarcinoma with a high probability of invasive and metastatic development.

The serodiagnosis was carried out at that time to reveal that the absorbance at 450 nm was 0.26. On June 3,2008, about 1 year after the extirpation, the serodiagnosis was carried out to find that the absorbance at 450 nm greatly decreased to 0.13.

0 Although any recurrent tumors were not found with the naked eye, an anticancer drug (INTERCAT) was administered once-weekly for 2 months to prevent recurrence. The serodiagnosis was carried out 2,4, and 6 weeks after the administration of the anticancer drug started to reveal that the absorbance at 450 nm was 0.09, 0.07 and 0.08, respectively. These results obtained in Canine Patient 11 confirmed that the

5 value becomes lower than that detected in a cancer-bearing state if tumors can be completely removed, as well as that the value does not increase if anticancer drug treatment successfully prevents cancer metastasis, and thus change in treated patients

2017239516 04 Oct 2017 can be followed. In addition, the diagnosis of recurrence can also be carried out as shown in Canine Patient 8, which confirms that the therapy monitoring can also be made possible.

[0134] (2)-8 Diagnosis of Malignancy of Recurrent Tumor

Canine Patient 12 (Chihuahua) underwent tumor extirpation on April 27, 2007. According to the pathological diagnosis using the extirpated tumor, this patient was suffering from ductal carcinoma originated from mammary ductal epithelium, i.e., malignant breast cancer. On June 29,2008, about 1 year thereafter, tumor was found again and extirpated. According to the pathological diagnosis using the extirpated tumor, although tumor cells which were originated from mammary ductal epithelium formed irregular glandular cavities and developed to reduplicate toward the lumens, the constituting cells had an almost uniformly eggshaped nucleus and atypism of the cells was low, and therefore the tumor was diagnosed as benign mammary adenocarcinoma. The serodiagnosis was carried out but the absorbance at 450 nm was 0.02, hardly detected. The results observed in Canine Patients 8 and 12 confirmed that the value of the serodiagnosis does not decrease or is sustained in cases where the recurrent tumor is malignant, and is hardly detected in cases where the tumor is benign.

[0135] (2)-9 Prognosis of Canine Patient Bearing Benign Tumor

Canine Patient 13 (Toy Poodle) underwent tumor extirpation on October 9, 2007. The pathological diagnosis using the extirpated tumor revealed that mammary epithelial cells and myoepithelial cells were both proliferated to form the

5 tumor, but that both of them did not show any malignant findings, and therefore this patient was diagnosed as benign mixed tumor. According to the serodiagnosis carried out at that time, the absorbance at 450 nm was 0.07, slightly detected. On

2017239516 04 Oct 2017

June 5,2008, 8 months thereafter, a blood sample was collected again and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0, not detected at all. Recurrence was not found clinically at that time. These results indicated that, even in the case where tumor is benign, complete removal of the tumor results in the decreased value of the serodiagnosis if a detectable value can be observed in cancer-bearing state, and hence prognosis can be attained.

[0136] (3) Diagnosis in Cats

Next, cancer-bearing cats and healthy cats were diagnosed. Using the above-described recombinant canine protein and anti-cat IgG antibody, the IgG antibody titer of feline serum which specifically reacts with the polypeptide was measured in the same manner as described above. As a secondary antibody, HRPconjugated anti-cat IgG antibody (PEROXIDASE-CONJUGATED GOAT IgG FRACTION TO CAT IgG (WHOLE MOLECULE): manufactured by CAPPEL

RESERCH REAGENTS) 8,000-fold diluted with the blocking solution was used. [0137]

Feline Patient 1 (Chinchilla) underwent tumor extirpation of mammary adenocarcinoma on August 17, 2005. The absorbance at 450 nm was 0.32. In Feline Patient 2 (Himalayan), which underwent extirpation of ductal carcinoma on

0 October 17, 2006, the absorbance at 450 nm was 0.18. On the other hand, the absorbance was not detected in healthy cats at all.

[0138]

Thus, similarly to dogs, the absorbance value was detected in samples from cats suffering from cancer, while the absorbance value was not detected at all in

5 samples from healthy cats. Hence, similarly to dogs, cancers in cats can also be detected by this method using a recombinant canine protein.

[0139]

2017239516 04 Oct 2017 (4) Diagnosis in Healthy Human

Using the above-described recombinant canine protein and anti-human IgG antibody above, the IgG antibody titer of healthy human serum which specifically reacts with the polypeptide was measured in the same manner as described above.

As a secondary antibody, HRP-conjugated anti-human IgG antibody (HRP-Goat Anti-Human IgG(H+L) Conjugate: manufactured by Zymed Laboratories) 10,000fold diluted with the blocking solution was used. As a positive control, an immobilized ovalbumin antigen prepared by immobilizing 50 pg/ml ovalbumin in phosphate buffered saline on a solid phase was used. As a result, in Healthy Human

1, the absorbance at 450 nm observed on an ovalbumin antigen was 0.25, while the absorbance at 450 nm observed on the recombinant protein was 0, not detected at all. Similarly, in Healthy Human 2, the absorbance at 450 nm observed on an ovalbumin antigen was 0.18, while the absorbance at 450 nm observed on the recombinant protein was 0, not detected at all.

[0140]

Example A-4: Cancer Diagnosis Using Recombinant Human Protein

Using the recombinant human protein prepared in Example A-2, the IgG antibody titer of human, canine and feline sera which react with the protein was measured in the same manner as in Example A-3.

[0141]

The diagnosis was carried out using healthy human serum. In the same manner as in Example A-3 (4), ovalbumin antigen was used as a positive control. As a result, the absorbance value was detected in the case where ovalbumin was immobilized on a solid phase, while the absorbance value was hardly detected in the

5 case where a human calmegin protein was immobilized on a solid phase.

[0142]

Similarly, in healthy dogs and cats, the absorbance at 450 nm was hardly

2017239516 04 Oct 2017 detected in the case where the protein was immobilized on a solid phase.

[0143]

On the other hand, Canine Patient 10 (Shih Tzu) underwent extirpation of mammary adenocarcinoma on June 21, 2007. According to the pathological diagnosis using the extirpated tissue, the mammary gland tissue contained highly atypical, invasive cells, and grew to form adenomatous hyperplasia showing large and small massive structures. Hence, this patient was diagnosed as malignant tumor. In this Canine Patient 10, the absorbance at 450 nm was 0.29. The malignancy diagnosis was carried out using additional 310 serum samples which had been diagnosed as malignant based on the pathological diagnosis. As a result, by diagnosing a sample showing twice the average value of healthy canine samples as malignant, 189 samples i.e. 60.8% of the malignant cases could be successfully diagnosed as malignant. Moreover, in Feline Patient 3 (Mixed Breed), which underwent extirpation of mammary adenocarcinoma on April 3, 2007, the absorbance 15 at 450 nm was 0.14.

[0144]

The above-described results indicate that the diagnosis can be similarly attained in humans, dogs and cats even by using the recombinant human protein. [0145]

0 Furthermore, pleural effusion and ascites samples collected from terminal cancer dogs were subjected to the diagnosis using the recombinant human protein in the same manner as the recombinant canine protein. As a result, values similar to those detected in serum samples could be detected, and hence cancer diagnosis could be successfully attained.

[0146]

Example A-5: Cancer Diagnosis by Measuring Antigen Polypeptide (1)

Mice and rabbits were immunized with the recombinant canine polypeptide

2017239516 04 Oct 2017 prepared in Example A-2 to obtain an antibody specific to this antigen. Using this polyclonal antibody, detection of the antigen polypeptide per se contained in the serum from cancer bearing living body was carried out by sandwich ELISA, Using anti-mouse IgG antibody, the amount of the protein in the serum which specifically reacts with the prepared polyclonal antibody specific to the protein was measured by sandwich ELISA.

[0147]

As for immobilization of a primary antibody on a solid phase, 100 pL/well of the rabbit antiserum 20-fold diluted with phosphate buffered saline was added to a

96-well Immobilizer Amino plate (manufactured by Nunc), and the plate was shaken at room temperature for 2 hours. As for blocking, 100 pL/well of 50 mM sodium bicarbonate buffer (pH 8.3) containing 0.5% BSA (bovine serum albumin, manufactured by Sigma Aldrich Japan) (hereinafter referred to as blocking solution) was added to the plate, and the plate was shaken at room temperature for 1 hour. To the plate, 100 pL/well of the serum from cancer-bearing body diluted with the blocking solution was added, and the plate was shaken at room temperature for 3 hours to allow the reaction to proceed. As for the diluted serum, a 10-fold serial dilution ranging 10 to 1,000-fold was prepared. After washing the wells 3 times with phosphate buffered saline containing 0.05% Tween20 (manufactured by Wako

0 Pure Chemicals) (hereinafter referred to as PBS-T), 100 pL/well of mouse antiserum 200-fold diluted with the blocking solution was added thereto, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 pL/well of HRP-conjugated mouse IgG antibody (Stabilized Goat Anti Mouse HRP conjugated : manufactured by PIERCE)

5 2,000-fold diluted with the blocking solution was added thereto as a tertiary antibody, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 μΙ/well of a HRP

2017239516 04 Oct 2017 substrate, TMB (1-Step Turbo TMB (tetramethylbenztdine), manufactured by PIERCE), was added thereto, and the enzyme-substrate reaction was allowed to proceed at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding 100 μΐ/well of 0.5 M sulfuric acid solution (manufactured by Sigma

Aldrich Japan), and then the absorbance was measured at 450 nm with a microplate reader. As a control, a plate on which the rabbit antiserum was not immobilized and a plate with which serum from a cancer-bearing body was not reacted were measured in the same manner as described above.

[0148]

Asa result, the polypeptide was detected in cancer-bearing dogs and cats suffering from cutaneous leiomyosarcoma, breast cancer, malignant melanoma or the like, while the polypeptide was not detected in healthy dogs, healthy cats and healthy humans. Hence, cancers could also be diagnosed by this method in which the antigen polypeptide was detected with an antibody prepared by using the recombinant canine polypeptide as an immunogen.

[0149]

Example A-6: Cancer Diagnosis by Measuring Antigen Polypeptide (2)

Mice and rabbits were immunized with the recombinant human protein prepared in Example A-2 to obtain an antibody specific to this antigen. In the same 2 0 manner as in Example 5, detection of the antigen polypeptide per se contained in the serum from cancer-bearing body was carried out by sandwich ELISA using this polyclonal antibody.

[0150]

As a result, the polypeptide was detected in cancer-bearing dogs and cats

5 suffering from cutaneous leiomyosarcoma, breast cancer, malignant melanoma or the like, while the polypeptide was not detected in healthy dogs, healthy cats and healthy humans. Hence, cancers could also be diagnosed by this method in which the

2017239516 04 Oct 2017 antigen polypeptide was detected with an antibody prepared by using the recombinant human polypeptide as an immunogen.

[0151]

Example B-l: Acquisition of Novel Cancer Antigen Protein by SEREX Method (1) Preparation of cDNA Library

Total RNA was prepared from testis tissue of a healthy dog by the Acid guanidium-Phenol-Chloroform method, and poly(A) RNA was purified using Oligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co., Ltd.) in accordance with the protocol attached to the kit.

[0152]

Using the obtained mRNA (5 pg), a dog testis cDNA phage library was synthesized. Preparation of the cDNA phage library was carried out using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit (manufactured by STRATAGENE) in accordance with the protocols attached to the kits. The size of the prepared cDNA phage library was 1.3 x 10⁶ pfu/ml.

[0153] (2) Screening of cDNA Library with Serum

Using the dog testis-derived cDNA phage library prepared as described above, immunoscreening was carried out. More particularly, host E. coli cells (XL 1-Blue

0 MRF') were infected with the library such that 2,340 clones should appear on an

NZY agarose plate having the size of Φ90 * 15 mm, and cultured at 42°C for 3 to 4 hours to allow the phage to form plaques. The plate was covered with nitrocellulose membrane (Hybond C Extra: manufactured by GE Healthcare Bio-Science) impregnated with IPTG (isopropyl-P-D-thiogalactoside) at 37°C for 4 hours to induce 2 5 and express proteins, which were thus transferred to the membrane. Subsequently, the membrane was recovered and soaked in TBS (10 mM Tris-HCl, 150 mM NaCl;

pH 7.5) containing 0,5% non-fat dry milk, followed by shaking at 4°C overnight to

2017239516 04 Oct 2017 suppress non-specific reactions. This filter was allowed to react with 500-fold diluted canine patient serum at room temperature for 2 to 3 hours.

[0154]

As the above-described canine patient serum, serum collected from canine patients suffering from tumor proximal to the anus was used . The serum was stored at -80°C and pretreated immediately before use. The method of the pretreatment of the serum was as follows. That is, host E. coli cells (XL 1 -Blue MRF) were infected with λ ZAP Express phage to which no foreign gene was inserted, and then cultured on NZY plate medium at 37°C overnight. Subsequently, the buffer of 0.2 M NaHCOj, pH 8.3 containing 0.5 M NaCl was added to the plate, and the plate was left to stand at 4°C for 15 hours, followed by collecting the supernatant as an E. colilpbage extract. Thereafter, the collected E. coliAh^ extract was allowed to flow through an NHS column (manufactured by GE Healthcare Bio-Science) to immobilize proteins derived from the E. colihphagQ thereon. The serum from the canine patients was allowed to flow through and react with this protein-immobilized column to remove antibodies adsorbed on E. coli and/or the phage. The serum fraction that passed through the column was 500-fold diluted with TBS containing 0.5% non-fat dry milk, and the resulting diluent was used as the material for the immuno screening.

[0155]

The membrane on which the thus treated serum and the above-described fusion protein were blotted was washed 4 times with TBS-T (0.05% Tween 20/TBS), and allowed to react with goat anti-dog IgG (Goat anti Dog IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5,000-fold diluted with TBS containing 25 0.5% non-fat dry milk as a secondary antibody at room temperature for 1 hour, followed by detection by the enzyme coloring reaction using the NBT/BCIP reaction solution (manufactured by Roche). Colonies at positions where a positive coloring

2017239516 04 Oct 2017 reaction was observed were recovered from the NZY agarose plate having the size of Φ90 x 15 mm, and dissolved in 500 μΐ of SM buffer (100 mM NaCl, 10 mM MgClSO₄, 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). The screening was repeated as a second and third screening in the same manner as described above until a single coloring reaction-positive colony was obtained, thereby isolating one positive clone after screening of 30,940 phage clones reactive with IgG in the serum.

[0156] (3) Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-described method to a base sequence analysis, an operation of conversion of the phage vector to a plasmid vector was carried out. More particularly, 200 μΐ of a solution prepared to contain a host E. coli (XL 1-Blue MRF) such that the absorbance OD^oo should be 1.0 was mixed with 100 μΐ of a purified phage solution and further with 1 μΐ of Ex Assist helper phage (manufactured by STRATAGENE), and the reaction was allowed to proceed at 37°C for 15 minutes. To the reaction mixture, 3 ml of LB medium was added, and the mixture was cultured at 37°C for 2.5 to 3 hours, followed by immediate incubation in a water bath at 70°C for 20 minutes. The mixture was then centrifuged at 4°C at 1,000 xg for 15 minutes, and the supernatant was recovered as a phagemid solution. Subsequently, 200 μΐ of a solution prepared to contain a

0 phagemid host E. coli (SOLR) such that the absorbance ODryjo should be 1.0 was mixed with 10 μΐ of a purified phage solution, and the reaction was allowed to proceed at 37°C for 15 minutes. Thereafter, 50 μΐ of the reaction mixture was plated on ampicillin (final concentration: 50 pg/ml)-containing LB agar medium, and cultured at 37°C overnight. A single colony of transformed SOLR was recovered

5 and cultured in ampicillin (final concentration: 50 pg/ml)-containing LB medium at 37°C, followed by purification of plasmid DNA having an insert of interest using QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

2017239516 04 Oct 2017 [0157]

The purified plasmid was subjected to an analysis of the entire sequence of the insert by the primer walking method using the T3 primer described in SEQ ID NO: 5 and the T7 primer described in SEQ ID NO :6, By this sequence analysis, the gene sequence described in SEQ ID NO: 15 was obtained. Using the base sequence and the amino acid sequence of this gene, homology search against known genes was carried out using a homology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), As a result, it was revealed that the obtained gene is the calmegin gene. The human homologous factor of the canine calmegin gene was human calmegin (homology: base sequence, 90%; amino acid sequence, 89%). The base sequence of human calmegin is shown in SEQ ID NO: 17, and the amino acid sequence thereof is shown in SEQ ID NO: 18.

[0158] (4) Analysis of Expression in Each Tissue

The expression of the gene, which was obtained by the above-described method, in normal tissues and various cell lines of dog and human were investigated by the RT-PCR (Reverse Transcription-PCR) method, The reverse transcription reaction was carried out as follows. That is, total RNA was extracted from 50 to 100 mg of each tissue or 5 to 10 * 10⁶ cells of each cell line using TRIZOL reagent

0 (manufactured by Invitrogen) in accordance with the protocol attached to the kit. Using this total RNA, cDNA was synthesized by Superscript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) in accordance with the protocol attached to the kit. As the cDNAs from human normal tissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA (manufactured by Invitrogen), QUICK2 5 Clone cDNA (manufactured by CLONTECH) and Large-Insert cDNA Library (manufactured by CLONTECH) were used. The PCR reactions were carried out as follows using primers (described in SEQ ID NOs:19 and 20) specific to the obtained

2017239516 04 Oct 2017 gene. That is, respective reagents and the attached buffer were mixed such that the mixture should contain 0.25 μΐ of the sample prepared by the reverse transcription reaction, 2 μΜ each of the above primers, 0.2 mM each of dNTP and 0.65 U of ExTaq polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 25 μΐ, and the reaction was carried out with 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute using Thermal Cycler (manufactured by BIO RAD). The above-described gene-specific primers were those which amplify the regions of the 755th to 1318th bases of the base sequence of SEQ ID NO: 15 (canine calmegin gene) and the 795th to 1358th bases of the base sequence of SEQ ID NO: 17, and can be used for investigation of the expression of both the canine calmegin gene and the human calmegin gene. As a control for comparison, primers (described in SEQ ID NOs:9 and 10) specific to GAPDH were used simultaneously. As a result, as shown in Fig. 5, strong expression was observed in testis among the normal dog tissues, and on the other hand, strong expression was observed in canine tumor cell lines. Expression of the human calmegin gene was confirmed, as is the case with the canine calmegin gene, only in testis among the normal tissues, but the expression was detected in brain tumor, leukemia and esophagus cancer ceils among cancer cell lines. Thus, the human calmegin gene was also confirmed to be specifically expressed in testis and cancer cells.

[0159]

In Fig. 5, reference numeral 1 in the ordinate indicates the expression pattern of the calmegin gene, and reference numeral 2 indicates the expression pattern of the GAPDH gene as a control for comparison.

[0160]

5 Example B-2: Preparation of Canine and Human Calmegin Proteins (1) Preparation of Recombinant Protein

Based on the gene of SEQ ID NO:15 obtained in Example B-l, a recombinant

2017239516 04 Oct 2017 protein was prepared by the following method. Respective reagents and the attached buffer were mixed such that the mixture should contain 1 μΐ of the vector that was prepared from the phagemid solution obtained in Example B-l and was subjected to the sequence analysis, 0.4 μΜ each of two kinds of primers having

Ziamlll and E'coRI restriction sites (described in SEQ ID NOs:21 and 22), 0.2 mM dNTP and 1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 50 μΐ, and PCR was carried out with 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds and 72°C for 2 minutes using Thermal Cycler (manufactured by BIO RAD). Using the above-described two kinds of primers, the region encoding the entire amino acid sequence of SEQ ID NO: 16 is obtained.

After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 1.9 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0161]

The purified DNA fragment was ligated into a cloning vector pCR-Blunt (manufactured by Invitrogen). E, coli was transformed with the resulting ligation product, and plasmids were recovered thereafter, followed by confirming, by sequencing, that the amplified gene fragment matches the sequence of interest. The plasmid that matched the sequence of interest was treated with restriction enzymes

0 BmwHI and EcoRI and purified using QIAquick Gel Extraction Kit, followed by inserting the gene sequence of interest into an expression vector for E. coli, pET30a (manufactured by Novagen) that had been treated with EiwzHI and EcoRI. Usage of this vector enables production of a His-tag fusion recombinant protein. E. coli for expression, BL21 (DE3), was transformed with this plasmid, and expression of

5 the protein of interest was induced in E. coli with 1 mM IPTG.

[0162]

Based on the gene of SEQ ID NO: 17, a recombinant protein of the human

2017239516 04 Oct 2017 homologous gene was prepared by the following method. Respective reagents and the attached buffer were mixed such that the mixture should contain 1 μΐ of the cDNA prepared in Example B-l whose expression could be confirmed by the RTPCR method in various tissues/cells, 0.4 μΜ each of two kinds of primers having

EcoRI and Xhol restriction sites (described in SEQ ID NOs:23 and 24). 0.2 mM dNTP and 1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 50 μΐ, and PCR was carried out with 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds and 72°C for 2 minutes using Thermal Cycler (manufactured by BIO RAD). Using the above-described two kinds of primers, the region encoding the entire amino acid sequence of SEQ ID NO: 18 is obtained.

After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 1.9 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0163]

The purified DNA fragment was ligated into a cloning vector pCR-Blunt (manufactured by Invitrogen). E. coli was transformed with the resulting ligation product, and plasmids were recovered thereafter, followed by confirming, by sequencing, that the amplified gene fragment matches the sequence of interest. The plasmid that matched the sequence of interest was treated with restriction enzymes

0 Ef-oRI and Xhol and purified using QIAquick Gel Extraction Kit, followed by inserting the gene sequence of interest into an expression vector for E. coli, pET30a (manufactured by Novagen) that had been treated with EvoRI and Xhol. Usage of this vector enables production of a His-tag fusion recombinant protein. E. coli for expression, BL21 (DE3), was transformed with this plasmid, and expression of the

5 protein of interest was induced in E. coli with 1 mM IPTG.

[0164] (2) Purification of Recombinant Protein

2017239516 04 Oct 2017

The above-obtained recombinant E. coli cells that expressed SEQ ID NO: 15 and SEQ ID NO: 17, respectively, were cultured in kanamycin (final concentration: 30 pg/ml)-containing LB medium at 37°C until the absorbance at 600 nm reached about 0.7, and then IPTG was added thereto such that its final concentration should 5 be 1 mM, followed by culturing them at 37°C for 4 hours. Subsequently, the cells were collected by centrifugation at 4,800 rpm for 10 minutes. The pellet of the ceils was suspended in phosphate-buffered saline and further subjected to centrifugation at 4,800 rpm for 10 minutes to wash the cells.

[0165]

The obtained pellet of E. coli cells was suspended in 20 mM phosphate buffer (pH 7.0) and subjected to sonication on ice. The sonicated solution of E. coli was centrifuged at 6,000 rpm for 20 minutes to obtain the supernatant as the soluble fraction and the precipitate as the insoluble fraction.

[0166]

The soluble fraction was placed in an cation-exchange column (carrier: SP

Sepharose (trademark) Fast Flow (GE Health Care); column volume: 5mL; equilibration buffer: 20 mM phosphate buffer (pH 7.0)). The column was washed with 10 column volumes of 20 mM phosphate buffer (pH 7.0), and then elution was immediately carried out with density gradient of salt by 0.3 M-1.0 M sodium

0 chloride-containing 20 mM phosphate buffer (pH 7.0). Six column volumes of the eluted fraction was collected in each elution step.

[0167]

Among these eluted fractions, all the fractions eluted with 0.3 M sodium chloride-containing 20 mM phosphate buffer (pH 7.0) and the 1st fraction eluted with 25 1.0 M sodium chloride-containing 20 mM phosphate buffer (pH 7.0) were combined, and the resulting solution was subjected to additional purification by a secondary column.

2017239516 04 Oct 2017 [0168]

For the secondary column, a column carrier Bio gel HT Type II (BioRad) was used. The column volume was 5 mL. The column was equilibrated with 10 column volumes of 0.3 M sodium chloride-containing 20 mM phosphate buffer (pH

7.0), and the above-described eluted fractions were placed in the column. The fractions that were not adsorbed to the column was washed away with 10 column volumes of 0.3 M sodium chloride-containing 20 mM phosphate buffer (pH 7.0) and 0.1 M phosphate buffer (pH 7.0). Immediately thereafter, elution was carried out with 0.2 M phosphate buffer (pH 7.0). Six column volumes of the eluted fraction was collected in each elution step. Elution of the proteins of interest was confirmed by Coomassie staining carried out according to a conventional method. Based on the result, the eluted fractions were desalted and concentrated to obtain the material to be solid-phased for diagnosis.

[0169]

Example B-3: Cancer Diagnosis Using Canine Calmegin Protein (1) Cancer Diagnosis in Dogs

Blood samples were collected from 486 canine patients in which malignant or benign tumors were found and 6 healthy dogs, and sera were separated therefrom. Using the canine calmegin protein prepared in Example B-2 and anti-dog IgG

0 antibody, the IgG antibody titer of the sera which specifically react with the protein was measured by ELISA.

[θΠΟ]

As for immobilization of the prepared protein on a solid phase, 100 pL/well of a solution of the recombinant protein diluted to 50 pg/mL with phosphate buffered 2 5 saline was added to a 96-well Immobilizer Amino plate (manufactured by Nunc), and the plate was left to stand at 4°C overnight. As for blocking, 100 pL/well of 50 mM sodium bicarbonate buffer (pH 8.3) containing 0.5% BSA (bovine serum albumin,

2017239516 04 Oct 2017 manufactured by Sigma Aldrich Japan) (hereinafter referred to as blocking solution) was added to the plate, and the plate was shaken at room temperature for 1 hour. Serum was 1,000-fold diluted with the blocking solution, and and 100 pL/well of the diluted serum was added to the plate, followed by shaking the plate at room temperature for 3 hours to allow the reaction to proceed. After washing the wells 3 times with phosphate buffered saline containing 0.05% Tween20 (manufactured by Wako Pure Chemicals) (hereinafter referred to as PBS-T), 100 pL/well of HRPconjugated dog IgG antibody (Goat anti Dog IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 3,000-fold diluted with the blocking solution was added thereto, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed.. After washing the wells 3 times with PBS-T, 100 μΐ/well of a HRP substrate, TMB (1-Step Turbo TMB (tetramethylbenzidine), manufactured by PIERCE), was added thereto, and the enzyme-substrate reaction was allowed to proceed at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding 100 μΐ/well of 0.5 M sulfuric acid solution (manufactured by Sigma Aldrich Japan), and then the absorbance was measured at 450 nm with a microplate reader. As a control, a plate on which the prepared recombinant protein was not immobilized and a plate with which the serum from a cancer-bearing dog was not reacted were measured in the same manner as above.

[0171]

Among the total 486 samples used in the above-described cancer diagnosis,

311 samples were definitely diagnosed as malignant by pathological diagnosis using the extirpated tumor tissue.

[0172]

5 Specifically, the samples were diagnosed as cancer such as malignant melanoma; malignant mixed tumor; hepatocellular carcinoma; basal cell carcinoma; acanthomatous epulis; intraoral tumor; perianal adenocarcinoma; anal sac tumor; anal

2017239516 04 Oct 2017 sac apocrine carcinoma; Sertoli cell tumor; vulva cancer; sebaceous adenocarcinoma; sebaceous epithelioma; sebaceous adenoma; sweat gland carcinoma; intranasal adenocarcinoma; nasal adenocarcinoma; thyroid cancer; colon cancer; bronchial adenocarcinoma; adenocarcinoma; ductal carcinoma; mammary adenocarcinoma;

combined mammary adenocarcinoma; mammary gland malignant mixed tumor; intraductal papillary adenocarcinoma; fibrosarcoma; hemangiopericytoma; osteosarcoma; chondrosarcoma; soft tissue sarcoma; histiocytic sarcoma; myxosarcoma; undifferentiated sarcoma; lung cancer; mastocytoma; cutaneous leiomyoma; intra-abdominal leiomyoma; leiomyoma; squamous cell carcinoma;

chronic lymphocytic leukemia; lymphoma; gastrointestinal lymphoma; digestive organ lymphoma; small cell or medium cell lymphoma; adrenomedullary tumor; granulosa cell tumor; pheochromocytoma; bladder cancer (transitional cell carcinoma); suppurative inflammation; intra-abdominal liver tumor; liver cancer; plasmacytoma; malignant hemangiopericytoma; angiosarcoma; anal sac adenocarcinoma; oral cancer; metastatic malignant melanoma; amelanotic malignant melanoma; cutaneous malignant melanoma; malignant myoepithelioma; malignant seminoma; seminoma; adenocarcinoma of the large intestine; gastric adenocarcinoma; low-grade sebaceous carcinoma; ceruminous adenocarcinoma; apocrine carcinoma; poorly differentiated apocrine sweat gland carcinoma; malignant

0 fibrous histiocytoma; multiple myeloma; mesenchymal malignant tumor; liposarcoma; osteosarcoma; sarcoma of unknown origin; soft part sarcoma (spindle cell tumor); poorly differentiated sarcoma; synovial sarcoma; angiosarcoma; metastatic malignant epithelioma; tubular mammary adenocarcinoma; mammary ductal carcinoma; inflammatory breast cancer; germinoma; leukemia; invasive trichoepithelioma; medium cell lymphoma; multicentric lymphoma; osteosarcoma (mammary gland); mastocytoma (Patnaik II type); mastocytoma (Grade II); leiomyosarcoma or the like.

2017239516 04 Oct 2017 [0173]

As shown in Fig. Ί, sera from these cancer-bearing dogs showed a significantly high antibody titer against the recombinant protein. It was revealed that, by diagnosing a sample showing twice the average value of healthy canine samples as malignant, 177 samples i.e. 56.9% of the malignant cases could be successfully diagnosed as malignant. The details of these 177 cancer samples are as follows. It is noted that the following number of each cancer case is a cumulative total, as some samples contained multiple primaries.

Malignant melanoma, 10 cases; lymphoma, 10 cases; pheochromocytoma, 1 case;

granulosa cell tumor, 1 case; hepatocellular carcinoma, 4 cases; sweat gland carcinoma, 5 cases; angioma, 1 case; malignant testicular tumor, 7 cases; intraoral tumor, 4 cases; perianal adenocarcinoma, 11 cases; osteosarcoma, 4 cases; fibrosarcoma, 7 cases; chondrosarcoma, 2 case; mammary adenocarcinoma, 35 cases; combined mammary adenocarcinoma, 27 cases; lung cancer, 2 cases; sebaceous adenocarcinoma, 2 cases; nasal adenocarcinoma, 2 cases; mastocytoma, 25 cases; adrenomedullary tumor, 1 case; leiomyosarcoma, 1 case; squamous cell carcinoma, 5 cases; chronic lymphocytic leukemia, 1 case; germinoma, 1 case; malignant fibrous histiocytoma, 1 case; metastatic malignant epithelioma, 1 case; mammary ductal carcinoma, 1 case; angiosarcoma, 1 case; tubular mammary adenocarcinoma, 1 case;

0 invasive trichoepithelioma, 1 case; prostate cancer, 1 case; bronchial adenocarcinoma, 1 case.

[0174]

The above-described diagnostic method was also carried out using pleural effusion samples and ascites samples collected from terminal cancer dogs. As a

5 result, values similar to those detected in serum samples could be detected, and hence cancer diagnosis could be successfully attained.

[0175]

2017239516 04 Oct 2017

Furthermore, it was confirmed that diagnostic approaches such as diagnosis of cancers existing in an invisible part of the body, assessment of cancer stage and grade, follow-up of postoperative patients, diagnosis of recurrence and metastasis and the like can also be attained by applying the above-described diagnostic method. The 5 followings are several of the practical examples of the detailed diagnosis shown in

Fig. 8.

[0176] (2)-1 Diagnosis of Invisible Tumors

In Canine Patient 1 (Flat-Coated Retriever), any tumors were not found on

June 7,2007. But about 20 days later, on June 24, 2007, a pedunculated tumor with a diameter of 2 mm was found on the gum at the root of the canine tooth. The tumor was ligated at its pedunculated part and excised on the day it found. The absorbance at 450 nm observed before the tumor became visible with the naked eye was 0.31, which was significantly high and not so different from the absorbance at the time of finding tumor, 0.33. This result indicates that it is possible to diagnose cancers even in an invisible part such as an intraperitoneal part by the method of the present invention.

[0177]

Rise of the value was observed before the tumor became visible with the

0 naked eye, which is considered to have been a sign of tumor development. Thus, the method of the present invention is useful in medical examinations such as periodic health examination.

[0178]

Canine Patient 1 was again checked by the serodiagnosis 2 weeks after the

5 tumor excision. As a result, the absorbance at 450 nm was greatly reduced to 0,17.

Thus, it was also confirmed that the cancer antigen-expressing tumor which had caused the increased antibody titer was completely removed (see, (2)-4, Follow-Up

2017239516 04 Oct 2017 of Postoperative Patients).

[0179] (2)-2 Assessment of Stage of Cancer Progression

The stage of cancer progression is determined based on the size or depth of the tumor, how much the tumor exerts influence on the surrounding tissues, whether the tumor metastasizes or not, and the like. It was revealed herein that the detected value is higher than before if the metastasis occurs, i.e., the cancer has advanced. The following is another example of a stage assessment of a certain cancer case, which received anticancer drug therapy.

[0180]

Canine Patient 2 (Miniature Dachshund) visited the hospital with chief complaints of nausea and emaciation on February 21,2007, and two massive tumors were found in the abdominal cavity. This patient underwent tumor extirpation on February 23,2007. The swollen right kidney weighed 433 g. The neighboring 15 lymph node was well-vascularized and weighed 42 g. Based on the pathological diagnosis using the extirpated tissue, the patient was diagnosed as multicentric malignant lymphoma. It was said that there was a probability that the tumor cells would spread into other organs in the abdominal cavity, as a disseminated spread of tumor cells was observed in the adipose tissue. The anticancer drug administration 2 0 (Oncovin) was started postoperatively on March 1, 2007, and the serodiagnosis was carried out 3 times, i.e., on the day the administration was started, and 2 and 3 months thereafter. As a result, the absorbance at 450 nm was 0.18,0.16, and 0.14, respectively. The value had gradually decreased since the start of the administration, which confirmed that the anticancer drug took effect. Thus, it was confirmed that 2 5 cancer progression could be inhibited. Hence, the results in Canine Patient 2 confirmed that the stage of cancer progression can also be assessed. In addition, it was confirmed that the effect of anticancer drug therapy can also be assessed as

2017239516 04 Oct 2017 described above.

[0181] (2)-3 Assessment of Grade of Cancer Malignancy

Basaliomas include malignant type and benign type. Recently, according to the new WHO classification, malignant basaliomas are called basal cell carcinoma, and benign basaliomas are called trichoblastoma.

[0182]

Canine Patient 3 (Beagle) was diagnosed as basal cell carcinoma (malignant).

The serodiagnosis was carried out at the time of the surgery to find that the absorbance at 450 nm was 0.13. On the other hand, in the case of Canine Patient 4 (Mixed Breed) diagnosed as trichoblastoma (benign), the serodiagnosis carried out at the time of the surgery revealed that the absorbance at 450 nm was 0, not detected at all. Thus, even in the case of the same basaliomas, malignant basal cell carcinoma and benign trichoblastoma can be distinctively diagnosed.

[0183]

Next example is mammary gland tumors. Mammary gland tumors include malignant tumors such as mammary adenocarcinoma and mammary gland malignant mixed tumor, and benign mammary tumors which do not show malignant symptoms. Canine Patient 5 (Yorkie) underwent extirpation of mammary gland malignant mixed 2 0 tumor and mammary adenocarcinoma on May 17,2006. In general, the complete excision of mixed tumors in mammary gland is easy because they are poorly invasive to the surrounding tissues even if they are malignant, and thus the postoperative course of the patients is usually uneventful. However, Canine Patient 5 had been diagnosed as highly malignant tumor, because the pathological diagnosis using the

5 extirpated tissue revealed that some components of the specimen from Canine Patient showed an invasive nature. On the other hand, mammary adenocarcinoma is a highly invasive tumor which often recurs and metastasizes. Although invasion of

2017239516 04 Oct 2017 tumor cells was not observed in the specimen from Canine Patient 5, it had been pointed out that highly malignant components possibly proliferated in other region out of the specimen. Thus, the findings in the pathological diagnosis clearly taught that Canine Patient 5 was suffering from highly malignant mammary cancer. A blood sample was collected during the surgery and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0.57. On the other hand, Canine Patient 6 (Yorkshire Terrier) underwent extirpation of mammary tumor on January 28, 2007. According to the pathological diagnosis using the extirpated tissue, atypism of cells was low, and thus Canine Patient 6 was diagnoses as benign mastadenoma without malignant findings. A blood sample was collected during the surgery and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0. The results in the two cases above revealed that highly malignant tumors show a higher value than low malignant, benign tumors.

[0184] (2)-4 Follow-Up of Postoperative Patients

Canine Patient 7 (Shih Tzu) visited the hospital due to an intraoral tumor and underwent the extirpation on March 22,2007. The serodiagnosis was carried out then to find that the absorbance at 450 nm was 0.70. In addition, based on the pathological diagnosis using the extirpated tissue, Canine Patient 7 was diagnosed as

0 malignant acanthomatous epulis. This kind of tumor often recurs if excision is insufficient, though distant metastasis seldom happens. Thus, it is important whether the tumor can be completely excised by surgery or not. According to the follow-up on May 18,2007, the absorbance at 450 nm decreased to 0.47. The recurrence has not been found till August of 2007. Thus, it is considered that the

5 value obtained by the serodiagnosis became lower than that obtained at the time of surgery because the tumor could be completely excised from Canine Patient 7. [0185]

2017239516 04 Oct 2017 (2)-5 Diagnosis of Recurrence

Canine Patient 8 (Husky) underwent extirpation of mammary adenocarcinoma on May 8,2007. The serodiagnosis was carried out at the time of the surgery to find that the absorbance at 450 nm was 0.11. The pathological diagnosis using the extirpated tissue revealed that highly atypical epithelial cells proliferated and mainly formed ductal structures, and thus this patient was diagnosed as primary breast adenocarcinoma. It was said that the patient was at a high risk of recurrence or metastasis to lymph nodes or distant organs, as many cancer cells had already entered the lymph vessels at that time. On June 28,2007, about 1 -and-a-half-month after the surgery, metastasis was found at the same site. The value detected by the serodiagnosis increased to 0.12. Thus, it was confirmed that the value detected by the serodiagnosis was higher in the end of June than in the beginning of May because the tumor could not have been completely excised or recurrence would have occurred in Canine Patient 8.

[0186]

Canine Patient 9 (Sheltie) underwent extirpation of ductal carcinoma on October 24,2006. The serodiagnosis carried out at that time revealed that the absorbance at 450 nm was approximately 0, hardly detected. About 3 months later, on January 31,2007, this patient visited the hospital because of cancer recurrence

0 and underwent extirpation again. According to the pathological diagnosis using the extirpated tissue, many cancer cells having egg-shaped atypical nuclei invaded lymph vessels and metastasis was observed in the inguinal lymph node, and thus the patient was diagnosed as ductal carcinoma (breast cancer) with a probability of distant metastasis. The serodiagnosis was carried out at that time to find that the

5 absorbance at 450 nm increased to 0.10. Thus, similarly to above, it was revealed that the value of the serodiagnosis increased 3 months later because the tumor could not have been completely excised or recurrence of the tumor would have occurred in

2017239516 04 Oct 2017

Canine Patient 9.

[0187] (2)-6 Diagnosis of Metastasis

Canine Patient 10 (Scottish Terrier), repeatedly undergoing metastasis and recurrence, was diagnosed as mammary tumor in February of 2003; intraoral malignant melanoma in August of 2003; malignant melanoma of the lip in January of 2005; and as intraoral melanoma on April 13,2005, all of which were excised by surgery. This patient visited the hospital again on December 17,2006 for follow-up after the recurrence of intraoral melanoma in April of 2005, and the serodiagnosis was carried out at that time to find that the absorbance at 450 nm was 0.39. Half a year later, on June 20,2007, the patient again visited the hospital because of the hypertrophy of cervical and malar lymph nodes. In the case of lymphomas, hypertrophy of lymph nodes is systemically observed. Because Canine Patient 10 had only two swollen lymph nodes, this patient was clinically diagnosed as probable metastatic lymphoma. The diagnosis according to the present invention also revealed that it was a tumor which had metastasized from the tumor previously existed in this patient as the absorbance at 450 nm greatly increased to 0.80. [0188]

Canine Patient 11 (Shiba Inu) underwent extirpation of oral malignant

0 melanoma of the right lip on March 11,2006. This patient has a history of anticancer drug treatment (cyclophosphamide) from June 10 to September 26 in 2006, and has received BIREMO S, which contains organic germanium as a main ingredient, since May 23,2006. On March 20,2007, this patient underwent extirpation of a tumor which was considered to be metastasis from the tumor

5 mentioned above, and the serodiagnosis was carried out. As a result, the absorbance at 450 nm was 0.06. Based on the pathological diagnosis using the tissue extirpated at that time, Canine Patient 11 was diagnosed as metastatic malignant melanoma.

2017239516 04 Oct 2017

On June 27,2007, three months after the extirpation of metastatic melanoma, metastasis occurred in this patient again. The tumor which was extirpated on March 20,2007 existed in the right cervical part, and this time tumor occurred on the opposite side. As for the shape of the tumor, a black mass was formed similarly to the previous tumor. The tumor, having the size of 3.1 x 3.2 x 0.8 cm, was also clinically diagnosed as metastasis. The serodiagnosis was carried out again to find that the absorbance at 450 nm increased to 0.19, which indicated that it was metastatic tumor.

[0189] (2)-7 Therapy Monitoring

Canine Patient 11 (Miniature Dachshund) underwent tumor extirpation on April 19,2007. According to the pathological diagnosis using the extirpated tumor, the patient was suffering from moderately-malignant combined mammary adenocarcinoma with a high probability of invasive and metastatic development.

The serodiagnosis was carried out at that time to find that the absorbance at 450 nm was 0.30. On June 3, 2008, about 1 year after the extirpation, the serodiagnosis was carried out to find that the absorbance at 450 nm decreased to 0.25. Although any recurrent tumors were not found with the naked eye, an anticancer drug (INTERCAT) was administered once-weekly for 2 months to prevent recurrence.

0 The serodiagnosis was carried out 2,4, and 6 weeks after the administration of the anticancer drug started to reveal that the absorbance at 450 nm was 0.25,0.19 and 0.19, respectively. These results obtained in Canine Patient 11 confirmed that the value becomes lower than that detected in a cancer-bearing state if tumors can be completely removed, as well as that the value does not increase if anticancer drug

5 treatment successfully prevents cancer metastasis, and thus change in treated patients can be followed. In addition, the diagnosis of recurrence can also be carried out as shown in Canine Patient 8, which confirms that the therapy monitoring can also be

2017239516 04 Oct 2017 made possible.

[0190] (2)-8 Diagnosis of Malignancy of Recurrent Tumor

Canine Patient 12 (Chihuahua) underwent tumor extirpation on April 27,

2007. According to the pathological diagnosis using the extirpated tumor, this patient was suffering from ductal carcinoma originated from mammary ductal epithelium, i.e., malignant breast cancer. On June 29,2008, about 1 year thereafter, tumor was found again and extirpated. According to the pathological diagnosis using the extirpated tumor, although tumor cells which were originated from mammary ductal epithelium formed irregular glandular cavities and developed to reduplicate toward the lumens, the constituting cells had an almost uniformly eggshaped nucleus and atypism of the cells was low, and therefore the tumor was diagnosed as benign mammary adenocarcinoma. The serodiagnosis was carried out but the absorbance at 450 nm was 0, not detected at all. The results observed in

Canine Patients 8 and 12 revealed that the value of the serodiagnosis does not decrease or is sustained in cases where the recurrent tumor is malignant, and is not detected in cases where the tumor is benign.

Prognosis of Canine Patient Bearing Benign Tumor

Canine Patient 13 (Toy Poodle) underwent tumor extirpation on October 9,

According to the pathological diagnosis using the extirpated tumor, mammary epithelial cells and myoepithelial cells were both proliferated to form the tumor, but both of them did not show any malignant findings, and therefore it was diagnosed as benign mixed tumor. The serodiagnosis showed the result that the

5 absorbance at 450 nm was 0.13, slightly detected. On June 5,2008, 8 months thereafter, a blood sample was collected again and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0, not detected at all. Recurrence was not [0191] (2)-9

2007.

2017239516 04 Oct 2017 found clinically at that time. These results confirmed that, even in the case where tumor is benign, complete removal of the tumor results in the decreased value in serodiagnosis if a detectable value can be observed in cancer-bearing state, and thus prognosis can be attained.

[0192] (3) Diagnosis in Cats

Next, cancer-bearing cats and healthy cats were diagnosed. Using the above-described canine calmegin protein and anti-cat IgG antibody, the IgG antibody titer of feline serum which specifically reacts with the polypeptide was measured in the same manner as described above. As a secondary antibody, HRP-conjugated anti-cat IgG antibody (PEROXIDASE-CONJUGATED GOAT IgG FRACTION TO CAT IgG (WHOLE MOLECULE): manufactured by CAPPEL RESERCH REAGENTS) 8,000-fold diluted with the blocking solution was used.

[0193]

Feline Patient 1 (Chinchilla) underwent tumor extirpation of mammary adenocarcinoma on August 17, 2005. The absorbance at 450 nm was 0.22. In Feline Patient 2 (Himalayan), which underwent extirpation of ductal carcinoma on October 17,2006, the absorbance at 450 nm was 0.21. On the other hand, the absorbance was not detected in healthy cats at all.

[0194]

Thus, similarly to dogs, the absorbance value was detected in samples from cats suffering from cancer, while the absorbance value was not detected at all in samples from healthy cats. Hence, similarly to dogs, cancers in cats can also be diagnosed by this method using a canine calmegin protein.

[0195] (4) Diagnosis in Healthy Human

Using the above-described canine calmegin protein and anti-human IgG

2017239516 04 Oct 2017 antibody above, the IgG antibody titer of healthy human serum which specifically reacts with the protein was measured in the same manner as described above. As a secondary antibody, HRP-conjugated anti-human IgG antibody (HRP-Goat AntiHuman IgG(H+L) Conjugate: manufactured by Zymed Laboratories) 10,000-fold diluted with the blocking solution was used. As a positive control, an immobilized ovalbumin antigen prepared by immobilizing 50 pg/ml ovalbumin in phosphate buffered saline on a solid phase was used. As a result, in Healthy Human 1, the absorbance at 450 nm observed on an ovalbumin antigen was 0.25, while the absorbance at 450 nm observed on the recombinant protein was 0.03, hardly detected.

[0196]

Example B-4: Cancer Diagnosis Using Human Calmegin Protein

Using the human calmegin protein prepared in Example B-2, the IgG antibody titer of human, canine and feline sera which react with the protein was measured in the same manner as in Example B-3.

[0197]

The diagnosis was carried out using healthy human serum. In the same manner as in Example B-3 (4), ovalbumin antigen was used as a positive control. As a result, the absorbance value was detected in the case where ovalbumin was immobilized on a solid phase, while the absorbance value was hardly detected in the 2 0 case where a human calmegin protein was immobilized on a solid phase.

[0198]

Similarly, in healthy dogs and cats, the absorbance at 450 nm was hardly detected in the case where the protein was immobilized on a solid phase. [0199]

On the other hand, Canine Patient 12 (Shih Tzu) underwent extirpation of mammary adenocarcinoma on June 21,2007. According to the pathological diagnosis using the extirpated tissue, the mammary gland tissue contained highly

100

2017239516 04 Oct 2017 atypical, invasive cells, and grew to form adenomatous hyperplasia showing large and small massive structures. Hence, this patient was diagnosed as malignant tumor. In this Canine Patient 12, the absorbance at 450 run was 0.70. The malignancy diagnosis was carried out using additional 310 serum samples which had been diagnosed as malignant based on the pathological diagnosis. As a result, by diagnosing a sample showing twice the average value of healthy canine samples as malignant, 171 samples i.e. 55.0% of the malignant cases could be successfully diagnosed as malignant. Moreover, in Feline Patient 3 (Mixed Breed), which underwent extirpation of mammary adenocarcinoma on April 3, 2007, the absorbance 10 at 450 nm was 0.38.

[0200]

The above described results indicates that the diagnosis can be similarly attained in humans, dogs and cats even by using a human calmegin protein. [0201]

Furthermore, pleural effusion and ascites samples collected from terminal cancer dogs were subjected to the diagnosis using the recombinant human protein in the same manner as the recombinant canine protein. As a result, values similar to those detected in serum samples could be detected, and hence cancer diagnosis could be successfully attained.

[0202]

Example B-5: Cancer Diagnosis by Measuring Antigen Polypeptide (1)

Mice and rabbits were immunized with the recombinant canine protein prepared in Example B-2 to obtain an antibody specific to this antigen. Using this polyclonal antibody, detection of the antigen polypeptide per se contained in the

5 serum from cancer bearing living body was carried out by sandwich ELISA. Using anti-mouse IgG antibody, the amount of the protein in the serum which specifically reacts with the prepared polyclonal antibody specific to the protein was measured by

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2017239516 04 Oct 2017 sandwich ELISA.

[0203]

As for immobilization of a primary antibody on a solid phase, 100 pL/well of the rabbit antiserum 20-fold diluted with phosphate buffered saline was added to a

96-well Immobilizer Amino plate (manufactured by Nunc), and the plate was shaken at room temperature for 2 hours. As for blocking, 100 pL/well of 50 mM sodium bicarbonate buffer (pH 8.3) containing 0.5% BSA (bovine serum albumin, manufactured by Sigma Aldrich Japan) (hereinafter referred to as blocking solution) was added to the plate, and the plate was shaken at room temperature for 1 hour. To the plate, 100 pL/well of the serum from cancer-bearing body diluted with the blocking solution was added, and the plate was shaken at room temperature for 3 hours to allow the reaction to proceed. As for the diluted serum, a 1 Ο-fold serial dilution ranging 10 to 1,000-fold was prepared. After washing the wells 3 times with phosphate buffered saline containing 0.05% Tween20 (manufactured by Wako

Pure Chemicals) (hereinafter referred to as PBS-T), 100 pL/well of mouse antiserum 200-fold diluted with the blocking solution was added thereto, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 pL/well of HRP-conjugated mouse IgG antibody (Stabilized Goat Anti Mouse HRP conjugated : manufactured by PIERCE)

0 2000-fold diluted with the blocking solution was added thereto as a tertiary antibody, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 μΐ/well of a HRP substrate, TMB (l~Step Turbo TMB (tetramethylbenzidine), manufactured by PIERCE), was added thereto, and the enzyme-substrate reaction was allowed to

5 proceed at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding 100 μΐ/well of 0.5 M sulfuric acid solution (manufactured by Sigma Aldrich Japan), and then the absorbance was. measured at 450 nm with a microplate

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2017239516 04 Oct 2017 reader. As a control, a plate on which the rabbit antiserum was not immobilized and a plate with which serum from a cancer-bearing body was not reacted were measured in the same manner as described above.

[0204]

Asa result, the polypeptide was detected in cancer-bearing dogs and cats suffering from cutaneous leiomyosarcoma, breast cancer, malignant melanoma or the like, while the polypeptide was not detected in healthy dogs, healthy cats and healthy humans. Hence, cancers could also be diagnosed by this method in which the antigen polypeptide was detected with an antibody prepared by using the recombinant canine polypeptide as an immunogen.

[0205]

Example B-6: Cancer Diagnosis by Measuring Antigen Polypeptide (2)

Mice and rabbits were immunized with the recombinant human protein prepared in Example B-2 to obtain an antibody specific to this antigen. In the same manner as in Example B-5, detection of the antigen polypeptide per se contained in the serum from cancer-bearing body was carried out by sandwich ELISA using this polyclonal antibody.

[0206]

As a result, the polypeptide was detected in cancer-bearing dogs and cats

0 suffering from cutaneous leiomyosarcoma, breast cancer, malignant melanoma or the like, while the polypeptide was not detected in healthy dogs, healthy cats and healthy humans. Hence, cancers could also be diagnosed by this method in which the antigen polypeptide was detected with an antibody prepared by using the recombinant human polypeptide as an immunogen.

[0207]

Example C-l: Acquisition of Novel Cancer Antigen Protein by SEREX Method (1) Preparation of cDNA Library

103

2017239516 04 Oct 2017

Total RNA was prepared from testis tissue of a healthy dog by the Acid guanidium-PhenoI-Chloroform method, and poly(A) RNA was purified using Oligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co,, Ltd.) in accordance with the protocol attached to the kit.

[0208]

Using the obtained mRNA (5 pg), a dog testis cDNA phage library was synthesized. Preparation of the cDNA phage library was carried out using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit (manufactured by STRATAGENE) in accordance with the protocols attached to the kits. The size of the prepared cDNA phage library was 1.3 χ 10⁶ pfu/ml.

[0209] (2) Screening of cDNA Library with Serum

Using the dog testis-derived cDNA phage library prepared as described above, immunoscreening was carried out. More particularly, host E. coli cells (XL 1-Blue 15 MRF') were infected with the library such that 2,340 clones should appear on an

NZY agarose plate having the size of Φ90 χ 15 mm, and cultured at 42°C for 3 to 4 hours to allow the phage to form plaques. The plate was covered with nitrocellulose membrane (Hybond C Extra: manufactured by GE Healthcare Bio-Science) impregnated with IPTG (isopropyl-β-D-thiogalactoside) at 37°C for 4 hours to induce 2 0 and express proteins, which were thus transferred to the membrane. Subsequently, the membrane was recovered and soaked in TBS (10 mM Tris-HCl, 150 mM NaCl; pH 7.5) containing 0.5% non-fat dry milk, followed by shaking it at 4°C overnight to suppress non-specific reactions. This filter was allowed to react with 500-fold diluted canine patient serum at room temperature for 2 to 3 hours.

[0210]

As the above-described canine patient serum, serum collected from canine patients suffering from squamous cell carcinoma was used. The serum was stored

104

2017239516 04 Oct 2017 at -80°C and pretreated immediately before use. The method of the pretreatment of the serum was as follows. That is, host E. coli cells (XLl-Blue MRF') were infected with λ ZAP Express phage to which no foreign gene was inserted, and then cultured on NZY plate medium at 37°C overnight. Subsequently, the buffer of 0.2 M

NaHCCE, pH 8.3 containing 0.5 M NaCl was added to the plate, and the plate was left to stand at 4°C for 15 hours, followed by collecting the supernatant as an E. colihphagQ extract. Thereafter, the collected E. colilphagp extract was allowed to flow through an NHS column (manufactured by GE Healthcare Bio-Science) to immobilize proteins derived from the E. coZLphage thereon. The serum from the canine patients was allowed to flow through and react with this protein-immobilized column to remove antibodies adsorbed on E. coli and/or the phage. The serum fraction that passed through the column was 500-fold diluted with TBS containing 0.5% non-fat dry milk, and the resulting diluent was used as the material for the immunoscreening.

[0211]

The membrane on which the thus treated serum and the above-described fusion protein were blotted was washed 4 times with TBS-T (0.05% Tween 20/TBS), and allowed to react with goat anti-dog IgG (Goat anti Dog IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5,000-fold diluted with TBS containing 20 0.5% non-fat dry milk as a secondary antibody at room temperature for 1 hour, followed by detection by the enzyme coloring reaction using the NBT/BCIP reaction solution (manufactured by Roche). Colonies at positions where a positive coloring reaction was observed were recovered from the NZY agarose plate having the size of Φ90 x 15 mm, and dissolved in 500 μΐ of SM buffer (100 mM NaCl, 10 mM

5 MgClSCL, 50 mM Tris-HCl, 0.01 % gelatin; pH 7.5). The screening was repeated as a second and third screening in the same manner as described above until a single coloring reaction-positive colony was obtained, thereby isolating one positive clone

105

2017239516 04 Oct 2017 after screening of 30,940 phage clones reactive with IgG in the serum.

[0212] (3) Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-described method to a base sequence analysis, an operation of conversion of the phage vector to a plasmid vector was carried out. More particularly, 200 μΐ of a solution prepared to contain a host E. coli (XL 1-Blue MRF') such that the absorbance OD^oo should be 1.0 was mixed with 100 μΐ of a purified phage solution and further with 1 μΐ of ExAssist helper phage (manufactured by STRATAGENE), and the reaction was allowed to proceed at 37°C for 15 minutes. To the reaction mixture, 3 ml of LB medium was added, and the mixture was cultured at 37°C for 2.5 to 3 hours, followed by immediate incubation in a water bath at 70°C for 20 minutes. The mixture was then centrifuged at 4°C at 1,000 xg for 15 minutes, and the supernatant was recovered as a phagemid solution. Subsequently, 200 μΐ of a solution prepared to contain a phagemid host E. coli (SOLR) such that the absorbance OD^oo should be 1.0 was mixed with 10 μΐ of a purified phage solution, and the reaction was allowed to proceed at 37°C for 15 minutes. Thereafter, 50 μΐ of the reaction mixture was plated on ampicillin (final concentration: 50 pg/ml)-containing LB agar medium, and cultured at 37°C overnight. A single colony of transformed SOLR was recovered

0 and cultured in ampicillin (final concentration: 50 gg/ml)-containing LB medium at 37°C, followed by purification of plasmid DNA having an insert of interest using QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

[0213]

The purified plasmid was subjected to an analysis of the entire sequence of the insert by the primer walking method using the T3 primer described in SEQ ID NO:5 and the T7 primer described in SEQ ID NO:6. By this sequence analysis, the gene sequence described in SEQ ID NO:25 was obtained. Using the base sequence

106

2017239516 04 Oct 2017 and the amino acid sequence of this gene, homology search against known genes was carried out using a homology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, it was revealed that the obtained gene has 99% homology (which was calculated only in the overlapping region) to the registered CEP gene described in SEQ ID NO:41 in terms of base sequence and amino acid sequence, so that the gene was judged as the CEP gene. The obtained human homologous factor of the canine CEP was human CEP (homology to the CEP gene described in SEQ ID NO:25: base sequence, 87%; amino acid sequence, 84%). The base sequence of human CEP is shown in SEQ ID

NO:27, and the amino acid sequence thereof is shown in SEQ ID NO:28.

[0214] (4) Analysis of Expression in Each Tissue

The expression of the gene, which was obtained by the above-described method, in normal tissues and various cell lines of dog and human were investigated 15 by the RT-PCR (Reverse Transcription-PCR) method. The reverse transcription reaction was carried out as follows. That is, total RNA was extracted from 50 to 100 mg of each tissue or 5 to 10 * 10⁶ cells of each cell line using TRIZOL reagent (manufactured by Invitrogen) in accordance with the protocol attached to the kit. Using this total RNA, cDNA was synthesized by Superscript First-Strand Synthesis 2 0 System for RT-PCR (manufactured by Invitrogen) in accordance with the protocol attached to the kit. As the cDNAs from human normal tissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA (manufactured by Invitrogen), QUICKClone cDNA (manufactured by CLONTECH) and Large-Insert cDNA Library (manufactured by CLONTECH) were used. The PCR reactions were carried out as 2 5 follows using primers (described in SEQ ID NOs:29 and 30) specific to the obtained gene. That is, respective reagents and the attached buffer were mixed such that the mixture should contain 0.25 μΐ of the sample prepared by the reverse transcription

107

2017239516 04 Oct 2017 reaction, 2 μΜ each of the above primers, 0.2 mM each of dNTP and 0.65 U of ExTaq polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 25 μΐ, and the reaction was carried out with 30 cycles of 94°C for 30 seconds, 5 5 °C for 30 seconds and 72°C for 30 seconds using Thermal Cycler (manufactured by BIO

RAD). The above-described gene-specific primers were those which amplify the regions of the 4582nd to 5124th bases of the base sequences of SEQ ID NOs:25 and 41 (canine CEP gene) and the 4610th to 5152nd bases of the base sequence of SEQ ID NO:27 (human CEP gene), and can be used for investigation of the expression of both the canine CEP gene and the human CEP gene. As a control for comparison, primers (described in SEQ ID NOs:9 and 10) specific to GAPDH were used simultaneously. As a result, as shown in Fig. 9, strong expression of the canine . CEP gene was observed in testis among the normal dog tissues, and on the other hand, strong expression was observed in the canine breast cancer cell line. Expression of the human CEP gene was confirmed, as is the case with the canine

CEP gene, only in testis among the human normal tissues, but the expression was detected in brain tumor, leukemia and esophagus cancer cells among human cancer cell lines, and especially, strong expression was observed in the leukemia cell line. Thus, the human CEP gene was also confirmed to be specifically expressed in testis and cancer cells.

[0215]

In Fig. 9, reference numeral 1 in the ordinate indicates the expression pattern of the CEP gene, and reference numeral 2 indicates the expression pattern of the GAPDH gene as a control for comparison.

[0216]

5 Example C-2: Preparation of Polypeptides Derived from Canine and Human CEPs (1) Preparation of Recombinant Protein

Based on the gene of SEQ ID NO:25 obtained in Example C-l, a recombinant

108

2017239516 04 Oct 2017 protein was prepared by the following method. Respective reagents and the attached buffer were mixed such that the mixture should contain 1 μΐ of the vector that was prepared from the phagemid solution obtained in Example C-l and was subjected to the sequence analysis, 0.4 μΜ each of two kinds of primers having

BamHI and Sall restriction sites (described in SEQ ID NOs:31 and 32), 0.2 mM dNTP and 1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 50 μΐ, and PCR was carried out with 30 cycles of 98°C for 10 seconds, 55°C for 5 seconds and .72°C for 7 minutes using Thermal Cycler (manufactured by BIO RAD). Using the above-described two kinds of primers, a region encoding an amino acid region (SEQ ID NO:35) of 1514th to 2339th amino acids of SEQ ID NO :26 is obtained. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 2.5 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN). [0217]

In the same manner, PCR was carried out using two kinds of primers described in SEQ ID NOs:37 and 38 to obtain the region encoding the entire amino acid sequence of SEQ ID NO;26. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 7.0 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0218]

Moreover, PCR was carried out using two kinds of primers described in SEQ ID NOs:37 and 43 to obtain the region encoding the entire amino acid sequence of SEQ ID NO:42. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 7.8 kbp was

5 purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0219]

Each of the purified DNA fragments was ligated into a cloning vector pCR109

2017239516 04 Oct 2017

Blunt (manufactured by Invitrogen). E. coli was transformed with the resulting ligation product, and plasmids were recovered thereafter, followed by confirming, by sequencing, that the amplified gene fragment matches the sequence of interest. The plasmid that matched the sequence of interest was treated with restriction enzymes

BmnHI and Sall and purified using QIAquick Gel Extraction Kit, followed by inserting the gene sequence of interest into an expression vector for E. coli, pET30a (manufactured by Novagen) that had been treated with BmnHI and Sall. Usage of this vector enables production of a His-tag fusion recombinant protein. E. coli for expression, BL21 (DE3), was transformed with this plasmid, and expression of the 10 protein of interest was induced in E. coli with 1 mM IPTG.

[0220]

Further, based on the gene of SEQ ID NO:27, a recombinant protein of the human homologous gene was prepared by the following method. Respective reagents and the attached buffer were mixed such that the mixture should contain 1 15 μΐ of the cDNA prepared in Example C-l whose expression could be confirmed by the RT-PCR method in various tissues/cells, 0.4 μΜ each of two kinds of primers having BtwHI and Sall restriction sites (described in SEQ ID NOs:33 and 34), 0.2 mM dNTP and 1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 50 μΐ, and PCR was carried out with 30 cycles 20 of 98°C for 10 seconds, 55°C for 5 seconds and 72°C for 7 minutes using Thermal Cycler (manufactured by BIO RAD). Using the above-described two kinds of primers, a region encoding an amino acid region (SEQ ID NOG6) of 1513rd to 2325th amino acids of SEQ ID NO:28 is obtained. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of 2 5 about 2.5 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0221]

110

2017239516 04 Oct 2017

In the same manner, PCR was carried out using two kinds of primers described in SEQ ID NOs:39 and 40 to obtain the region encoding the entire amino acid sequence of SEQ ID NO:28. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 7.0 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0222]

Each of the purified DNA fragments was ligated into a cloning vector pCRBlunt (manufactured by Invitrogen). E. coli was transformed with the resulting ligation product, and plasmids were recovered thereafter, followed by confirming, by 10 sequencing, that the amplified gene fragment matches the sequence of interest. The plasmid that matched the sequence of interest was treated with restriction enzymes EcwiHI and Sall and purified using QIAquick Gel Extraction Kit, followed by inserting the gene sequence of interest into an expression vector for E. coli, pET30a (manufactured by Novagen) that had been treated with E<wIII and Sall. Usage of 15 this vector enables production of a His-tag fusion recombinant protein. E. coli for expression, BL21 (DE3), was transformed with this plasmid, and expression of the protein of interest was induced in E. coli with 1 mM IPTG.

[0223] (2) Purification of Recombinant Protein

0 The above-obtained recombinant E. coli cells that expressed a part of SEQ ID

NO:26 and a part of SEQ ID NO:28, respectively, were cultured in kanamycin (final concentration: 30 pg/ml)-containing LB medium at 37°C until the absorbance at 600 nm reached about 0.7, and then IPTG was added thereto such that its final concentration should be 1 mM, followed by culturing them at 30°C for 20 hours.

5 Subsequently, the cells were collected by centrifugation at 4,800 rpm for 10 minutes. The pellet of the cells was suspended in phosphate-buffered saline and further subjected to centrifugation at 4,800 rpm for 10 minutes to wash the cells.

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2017239516 04 Oct 2017 [0224]

The cells were suspended in phosphate-buffered saline and subjected to sonication on ice. The sonicated solution of E. coli was centrifuged at 7,000 rpm for 20 minutes to obtain the supernatant as the soluble fraction and the precipitate as 5 the insoluble fraction. The insoluble fraction was suspended in 4% Triton X-100 solution and the resulting suspension was centrifuged at 7,000 rpm for 20 minutes. This operation was repeated twice and an operation of removal of proteases was carried out. The residue was suspended in 8 M urea (manufactured by Sigma Aldrich Japan)-containing 10 mM Tris-HCl, 100 mM phosphate buffer (hereinafter 10 referred to as 8 M urea solution) and a protease inhibitor cocktail solution, and the resulting suspension was left to stand at 4°C for 15 hours to denature proteins. [0225]

Thereafter, the suspension was centrifuged at 7,000 rpm for 20 minutes, and the obtained soluble fraction was placed in a nickel chelate column prepared by a 15 conventional method (carrier: Chelating Sepharose (trademark) Fast Flow (GE Health Care); column volume: 5mL; equilibration buffer: 8M urea solution), followed by leaving it to stand at 4°C overnight. The supernatant was recovered by centrifugation of this column carrier at 1,500 rpm for 5 minutes, and the column carrier was suspended in phosphate-buffered saline followed by refilling the column 2 0 with the resulting suspension. The fraction that was not adsorbed to the column was washed away with 5 column volumes of 8 M urea solution, 10 column volumes of 0.5 M sodium chloride-containing 0.1 M acetate buffer (pH 5,0) and 10 mM imidazole-containing 20 mM phosphate buffer (pH 8.0), and elution was immediately carried out with a five-step density gradient of 100 mM-500 mM

5 imidazole. Five column volumes of the eluted fraction was collected in each elution step. Elution of the proteins of interest was confirmed by Coomassie staining carried out according to a conventional method. Based on the result, the eluted

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2017239516 04 Oct 2017 fractions were desalted and concentrated to obtain the material to be solid-phased for diagnosis.

[0226]

In the same manner, the recombinant E. coli cells that expressed the full5 length of SEQ ID NOs:26,28 and 42, respectively, were cultured and the proteins of interest were purified to obtain the material to be solid-phased for diagnosis.

[0227]

Example C-3: Cancer Diagnosis Using Polypeptide Derived from Canine CEP (1) Cancer Diagnosis in Dogs

Blood samples were collected from 486 canine patients in which malignant or benign tumors were found and 6 healthy dogs, and sera were separated therefrom. Using the partial polypeptide of canine CEP (SEQ ID NO:35; 1514th to 2339th amino acid region of SEQ ID NO :26) prepared in Example C-2 and anti-dog IgG antibody, the IgG antibody titer of the sera which specifically react with the polypeptide was measured by ELISA.

[0228]

As for immobilization of the prepared protein on a solid phase, 100 pL/well of a solution of the recombinant protein diluted to 50 pg/mL with phosphate buffered saline was added to a 96-well Immobilizer Amino plate (manufactured by Nunc), and 2 0 the plate was left to stand at 4°C overnight. As for blocking, 100 pL/well of 50 mM sodium bicarbonate buffer (pH 8.3) containing 0.5% BSA (bovine serum albumin, manufactured by Sigma Aldrich Japan) (hereinafter referred to as blocking solution) was added to the plate, and the plate was shaken at room temperature for 1 hour. Serum sample was 500-fold diluted with the blocking solution, and 100 pL/well of 2 5 the diluted serum was added to the plate, followed by shaking the plate at room temperature for 3 hours to allow the reaction to proceed. After washing the wells 3 times with phosphate buffered saline containing 0.05% Tween20 (manufactured by

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2017239516 04 Oct 2017

Wako Pure Chemicals) (hereinafter referred to as PBS-T), 100 pL/well of HRPconjugated dog IgG antibody (Goat anti Dog IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 3,000-fold diluted with the blocking solution was added thereto, and the plate was shaken at room temperature for 1 hour to allow the reaction 5 to proceed.. After washing the wells 3 times with PBS-T, 100 μΐ/well of a HRP substrate, TMB (1-Step Turbo TMB (tetramethylbenzidine), manufactured by PIERCE), was added thereto, and the enzyme-substrate reaction was allowed to proceed at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding 100 μΙ/well of 0.5 M sulfuric acid solution (manufactured by Sigma

Aldrich Japan), and then the absorbance was measured at 450 nm with a microplate reader. As a control, a plate on which the prepared recombinant protein was not immobilized and a plate with which the serum from a cancer-bearing dog was not reacted were measured in the same manner as above.

[0229]

Among the total 486 samples used in the above-described cancer diagnosis,

311 samples were definitely diagnosed as malignant by pathological diagnosis using the extirpated tumor tissue.

[0230]

Specifically, the samples were diagnosed as cancer such as malignant

0 melanoma; malignant mixed tumor; hepatocellular carcinoma; basal cell carcinoma; acanthomatous epulis; intraoral tumor; perianal adenocarcinoma; anal sac tumor; anal sac apocrine carcinoma; Sertoli cell tumor; vulva cancer; sebaceous adenocarcinoma; sebaceous epithelioma; sebaceous adenoma; sweat gland carcinoma; intranasal adenocarcinoma; nasal adenocarcinoma; thyroid cancer; colon cancer; bronchial

5 adenocarcinoma; adenocarcinoma; ductal carcinoma; mammary adenocarcinoma; combined mammary adenocarcinoma; mammary gland malignant mixed tumor; intraductal papillary adenocarcinoma; fibrosarcoma; hemangiopericytoma;

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2017239516 04 Oct 2017 osteosarcoma; chondrosarcoma; soft tissue sarcoma; histiocytic sarcoma; myxosarcoma; undifferentiated sarcoma; lung cancer; mastocytoma; cutaneous leiomyoma; intra-abdominal leiomyoma; leiomyoma; squamous cell carcinoma; chronic lymphocytic leukemia; lymphoma; gastrointestinal lymphoma; digestive organ lymphoma; small cell or medium cell lymphoma; adrenomedullary tumor; granulosa cell tumor; pheochromocytoma; bladder cancer (transitional cell carcinoma); suppurative inflammation; intra-abdominal liver tumor; liver cancer; plasmacytoma; malignant hemangiopericytoma; angiosarcoma; anal sac adenocarcinoma; oral cancer; metastatic malignant melanoma; amelanotic malignant melanoma; cutaneous malignant melanoma; malignant myoepithelioma; malignant seminoma; seminoma; adenocarcinoma of the large intestine; gastric adenocarcinoma; low-grade sebaceous carcinoma; ceruminous adenocarcinoma; apocrine carcinoma; poorly differentiated apocrine sweat gland carcinoma; malignant fibrous histiocytoma; multiple myeloma; mesenchymal malignant tumor;

liposarcoma; osteosarcoma; sarcoma of unknown origin; soft part sarcoma (spindle cell tumor); poorly differentiated sarcoma; synovial sarcoma; angiosarcoma; metastatic malignant epithelioma; tubular mammary adenocarcinoma; mammary ductal carcinoma; inflammatory breast cancer; germinoma; leukemia; invasive trichoepithelioma; medium cell lymphoma; multicentric lymphoma; osteosarcoma

0 (mammary gland); mastocytoma (Patnaik II type); mastocytoma (Grade II); leiomyosarcoma or the like.

[0231]

As shown in Fig. 11, sera from these cancer-bearing dogs showed a significantly high antibody titer against the recombinant protein. It was revealed

5 that, by diagnosing a sample showing twice the average value of healthy canine samples as malignant, 197 samples i.e. 63.3% of the malignant cases could be successfully diagnosed as malignant. The details of these 197 cancer samples are as

115

2017239516 04 Oct 2017 follows. It is noted that the following number of each cancer case is a cumulative total, as some samples contained multiple primaries.

Malignant melanoma, 8 cases; lymphoma, 9 cases; pheochromocytoma, 1 case; suppurative inflammation, 1 case; granulosa cell tumor, 1 case; hepatocellular carcinoma, 5 cases; angioma, 1 case; malignant testicular tumor, 6 cases; intraoral tumor, 5 cases; perianal adenocarcinoma, 12 cases; osteosarcoma, 4 cases; fibrosarcoma, 8 cases; ductal carcinoma, 10 cases; chondrosarcoma, 2 cases; mammary adenocarcinoma, 35 cases; combined mammary adenocarcinoma, 24 cases; lung cancer, 2 cases; sebaceous adenocarcinoma, 2 cases; nasal adenocarcinoma, 2 cases; mastocytoma, 24 cases; adrenomedullary tumor, 1 case; leiomyosarcoma, 1 case; squamous cell carcinoma, 4 cases; chronic lymphocytic leukemia, 1 case; undifferentiated sarcoma, 1 case; malignant mixed tumor, 1 case; tumor in the posterior segment of the left lobe of the lung, 1 case; tumor in the right infra-axillary region, 1 case; tumor in the elbow of the right forelimb, 1 case; bladder cancer (transitional cell carcinoma), 1 case; metastatic malignant melanoma, 3 cases; amelanotic malignant melanoma, 1 case; adenocarcinoma of the large intestine, 1 case; plasmacytoma, 1 case; histiocytic sarcoma, 1 case; liposarcoma, 1 case; poorly differentiated sarcoma, 1 case; synovial sarcoma, 1 case; malignant hemangiopericytoma, 1 case; apocrine sweat gland carcinoma, 3 cases; bronchial adenocarcinoma, 1 case.

[0232]

The above-described diagnostic method was also carried out using pleural effusion samples and ascites samples collected from terminal cancer dogs. As a result, values similar to those detected in serum samples could be detected, and hence 2 5 cancer diagnosis could be successfully attained.

[0233]

Furthermore, it was confirmed that diagnostic approaches such as diagnosis of

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2017239516 04 Oct 2017 cancers existing in an invisible part of the body, assessment of cancer stage and grade, follow-up of postoperative patients, diagnosis of recurrence and metastasis and the like can also be attained by applying the above-described diagnostic method. The followings are several of the practical examples of the detailed diagnosis shown in

Fig, 12.

[0234] (2)-1 Diagnosis of Invisible Tumors

In Canine Patient 1 (Flat-Coated Retriever), any tumors were not found on

June 7,2007. But about 20 days later, on June 24,2007, a pedunculated tumor with .10 a diameter of 2 mm was found on the gum at the root of the canine tooth. The tumor was ligated at its pedunculated part and excised on the day if found. The absorbance at 450 nm observed before the tumor became visible with the naked eye was 0.41, which was significantly high and not so different from the absorbance at the time of finding tumor, 0.43. The result indicates that it is possible to diagnose cancers even in an invisible part such as an intraperitoneal part by the method of the present invention.

[0235]

Rise of the value was observed before the tumor became visible with the naked eye, which is considered to have been a sign of tumor development. Thus,

0 the method of the present invention is useful in medical examinations such as periodic health examination.

[0236]

Canine Patient 1 was again checked by the serodiagnosis 2 weeks after the tumor excision. As a result, the absorbance at 450 nm was greatly reduced to 0.06.

5 Thus, it was also confirmed that the cancer antigen-expressing tumor which had caused the increased antibody titer was completely removed (see, (2)-4, Follow-Up of Postoperative Patients).

117

2017239516 04 Oct 2017 [0237] (2)-2 Assessment of Stage of Cancer Progression

The stage of cancer progression is determined based on the size or depth of the tumor, how much the tumor exerts influence on the surrounding tissues, whether 5 the tumor metastasizes or not, and the like. It was revealed herein that the detected value is higher than before if the metastasis occurs, i.e., the cancer has advanced. The following is another example of a stage assessment of a certain cancer case, which received anticancer drug therapy.

[0238]

Canine Patient 2 (Miniature Dachshund) visited the hospital with chief complaints of nausea and emaciation on February 21,2007, and two massive tumors were found in the abdominal· cavity. This patient underwent tumor extirpation on February 23,2007. The swollen right kidney weighed 433 g. The neighboring lymph node was well-vascularized and weighed 42 g. Based on the pathological diagnosis using the extirpated tissue, the patient was diagnosed as multicentric malignant lymphoma. It was said that there was a probability that the tumor cells would spread into other organs in the abdominal cavity, as a disseminated spread of tumor cells was observed in the adipose tissue. The anticancer drug administration (Oncovin) was started postoperatively on March 1,2007, and the serodiagnosis was

0 carried out 3 times, i.e., on the day the administration was started, and 2 and 3 months thereafter. As a result, the absorbance at 450 nm was 0.15, 0.15, and 0.07, respectively. The value had gradually decreased since the start of the administration, which confirmed that the anticancer drug took effect. Thus, it was confirmed that cancer progression could be inhibited. Hence, the results in Canine Patient 2

5 confirmed that the stage of cancer progression can also be assessed. In addition, it was confirmed that the effect of anticancer drug therapy can also be assessed as described above.

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2017239516 04 Oct 2017 [0239] (2)-3 Assessment of Grade of Cancer Malignancy

Basaliomas include malignant type and benign type. Recently, according to the new WHO classification, malignant basaliomas are called basal cell carcinoma, and benign basaliomas are called trichoblastoma.

[0240]

Canine Patient 3 (Beagle) was diagnosed as basal cell carcinoma (malignant).

The serodiagnosis was carried out at the time of the surgery to find that the absorbance at 450 nm was 0.14. On the other hand, in Canine Patient 4 (Mixed

Breed) diagnosed as trichoblastoma (benign), the serodiagnosis carried out at the time of the surgery revealed that the absorbance at 450 nm was 0, not detected at all. Thus, even in the case of the same basaliomas, malignant basal cell carcinoma and benign trichoblastoma can be distinctively diagnosed.

[0241]

Next example is mammary gland tumors. Mammary gland tumors include malignant tumors such as mammary adenocarcinoma and mammary gland malignant mixed tumor, and benign mammary tumors which do not show malignant symptoms. Canine Patient 5 (Yorkie) underwent extirpation of mammary gland malignant mixed tumor and mammary adenocarcinoma on May 17,2006. In general, the complete

0 excision of mixed tumors in mammary gland is easy because they are poorly invasive to the surrounding tissues even if they are malignant, and thus the postoperative course of the patients is usually uneventful. However, Canine Patient 5 had been diagnosed as highly malignant tumor, because the pathological diagnosis using the extirpated tissue revealed that some components of the specimen from Canine Patient

5 showed an invasive nature. On the other hand, mammary adenocarcinoma is a highly invasive tumor which often recurs and metastasizes. Although invasion of the tumor cells was not observed in the specimen from Canine Patient 5, it had been

119

2017239516 04 Oct 2017 pointed out that highly malignant components possibly proliferated in other region out of the specimen. Thus, the findings in the pathological diagnosis clearly taught that Canine Patient 5 was suffering from highly malignant mammary cancer. A blood sample was collected during the surgery and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0.77. On the other hand, Canine Patient 6 (Yorkshire Terrier) underwent extirpation of mammary tumor on January 28, 2007.

According to the pathological diagnosis using the extirpated tissue, atypism of cells was low, and thus Canine Patient 6 was diagnosed as benign mastadenoma without malignant findings. A blood sample was collected during the surgery and the 10 serodiagnosis was carried out to find that the absorbance at 450 nm was 0. The results in the two cases above revealed that highly malignant tumors show a higher value than low malignant, benign tumors.

[0242] (2)-4 Follow-Up of Postoperative Patients

Canine Patient 7 (Mixed Breed) underwent extirpation of perianal adenoma in

August of 2003 and on August 9,2006. The tumor extirpated on August 9,2006 was clinically diagnosed as recurrence, because the similar tumor again occurred at the same site. The pathological diagnosis using the tissue extirpated at the second time revealed that tumor cells were highly invasive and atypical showing

0 anisokaryosis and dyskaryosis, and also that a lot of dividing nuclei were observed. Thus, the patient was diagnosed as malignant tumor. According to the diagnostic pathologist, it is necessary to pay attention to local recurrence or metastasis which would occur again. The serodiagnosis was carried out at that time. As a result, the absorbance at 450 nm was 0.43. On December 19,2006, about 4 months after the

5 surgery, the serodiagnosis was carried out again on the patient in the course of follow-up. As a result, the absorbance at 450 nm decreased to 0.32. Neither recurrence nor metastasis has been found till August of 2007. Thus, it is considered

120

2017239516 04 Oct 2017 that the value obtained in the serodiagnosis became lower than that obtained at the time of surgery because the tumor could be completely extirpated in Canine Patient 7. [0243] ¢2)-5 Diagnosis of Recurrence

Canine Patient 8 (Husky) underwent an extirpation of mammary adenocarcinoma on May 8, 2007. According to the serodiagnosis carried out at the time of the surgery, the absorbance at 450 nm was 0.09. The pathological diagnosis using the extirpated tissue revealed that highly atypical epithelial cells proliferated and mainly formed ductal structures, and thus this patient was diagnosed as primary breast adenocarcinoma. It was said that the patient was at a high risk of recurrence or metastasis to lymph nodes or distant organs, as many cancer cells had already entered the lymph vessels at that time. On June 28, 2007, about 1-and-a-half month after the surgery, metastasis was found at the same site. The serodiagnosis was carried out at that time to find that the value increased to 0.10. Thus, it was confirmed that the value detected by the serodiagnosis was higher in the end of June than in the beginning of May because the tumor could not have been completely excised or recurrence would have occurred in Canine Patient 8.

[0244]

Canine Patient 9 (Sheltie) underwent extirpation of ductal carcinoma on

0 October 24, 2006. The serodiagnosis was carried out at that time. As a result, the absorbance at 450 nm was 0.02. About 3 months later, on January 31, 2007, this patient visited the hospital because of cancer recurrence and underwent extirpation again. According to the pathological diagnosis using the extirpated tissue, many cancer cells having egg-shaped atypical nuclei invaded lymph vessels and metastasis

5 was observed in the inguinal lymph node, and thus the patient was diagnosed as ductal carcinoma (breast cancer) with a probability of distant metastasis. The serodiagnosis was carried out at that time to find that the absorbance at 450 nm

121

2017239516 04 Oct 2017 increased to 0.09. Thus, similarly to above, it was confirmed that the value of the serodiagnosis increased 3 months later because the tumor could not have been completely excised or recurrence of the tumor would have occurred in Canine Patient 9.

[0245] (2)-6 Diagnosis of Metastasis

Canine Patient 10 (Scottish Terrier), repeatedly undergoing metastasis and recurrence, was diagnosed as mammary tumor in February of 2003; intraoral malignant melanoma in August of 2003; malignant melanoma of the lip in January of

2005; and as intraoral melanoma on April 13,2005, all of which were excised by surgery. This patient visited the hospital again on December 17,2006 for follow-up after the recurrence of intraoral melanoma in April of 2005, and the serodiagnosis was carried out at that time. As a result, the absorbance at 450 nm was 0.42. Half a year later, on June 20, 2007, the patient again visited the hospital because of the hypertrophy of cervical and malar lymph nodes. In the case of lymphomas, hypertrophy of lymph nodes is systemically observed. Because Canine Patient 10 had only two swollen lymph nodes, this patient was clinically diagnosed as probable metastatic lymphoma. The diagnosis according to the present invention also revealed that it was a metastatic tumor from one which had previously existed in this

0 patient, as the absorbance at 450 nm greatly increased to 0.91.

[0246] (2)-7 Therapy Monitoring

Canine Patient 12 (Mixed Breed) underwent tumor extirpation on July 27, 2007. The pathological diagnosis using the extirpated tumor revealed that breast

5 cancer grew continuously in the mammary ducts. Thus, this patient was diagnosed as ductal carcinoma. According to the serodiagnosis carried out at that time, the absorbance at 450 nm was 0.24. Cancer recurrence has not been found up to this

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2017239516 04 Oct 2017 time, i.e. 13 months after the extirpation. The serodiagnosis was again carried out on September 3, 2007, about 1 month after the extirpation; October 12,2007,2 months after the extirpation; and on June 1,2008, 10 month after the extirpation. As a result, the absorbance at 450 nm was 0.18, 0.18 and 0.12, respectively.

[0247]

These results obtained in Canine Patient 12 confirmed that the value becomes lower than that detected in a cancer-bearing state if tumors can be completely removed, as well as that the value does not increase unless cancer recurs, and thus change in treated patients can be followed. In addition, the diagnosis of recurrence can also be carried out as shown in Canine Patient 8, which confirms that the therapy monitoring can also be made possible.

[0248] (2)-8 Diagnosis of Malignancy of Recurrent Tumor

Canine Patient 13 (Golden Retriever) underwent tumor extirpation on May 1,

2005. The pathological diagnosis using the extirpated tumor revealed that the tumor in this patient was malignant neoplastic lesion originated from mammary ductal epithelium, i.e., malignant mammary ductal carcinoma and malignant papillary carcinoma continuously growing through the mammary ducts. On June 28, 2008, about 3 years thereafter, tumor was found again and thus extirpation was carried out.

0 The pathological diagnosis using the extirpated tumor revealed that nothing but severe infiltration of inflammatory cells such as neutrophils, macrophages, plasma cells and the like could be observed around surgical sutures under the skin which was considered to be the previous surgical wound, and thus the patient was diagnosed as having no neoplastic lesions. According to the serodiagnosis carried out at that time,

5 the absorbance at 450 nm was 0, not detected at all. The results observed in Canine Patients 8, 9 and 13 indicated that the value of the serodiagnosis does not decrease or is sustained in cases where the recurrent tumor is malignant, and is not detected in

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2017239516 04 Oct 2017 cases where the tumor is benign.

[0249] (2) -9 Prognosis of Canine Patient Bearing Benign Tumor

Canine Patient 14 (Toy Poodle) underwent tumor extirpation on October 9,

2007. The pathological diagnosis using the extirpated tumor revealed that mammary epithelial cells and myoepithelial cells were both proliferated to form the tumor, but that both of them did not show any malignant findings, and therefore this patient was diagnosed as benign mixed tumor. According to the serodiagnosis carried out at that time, the absorbance at 450 nm was 0.05, slightly detected. On

June 5,2008, 8 months thereafter, a blood sample was collected again and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0, not detected at all. Recurrence was not found clinically at that time. These results indicated that, even in the case where tumor is benign, complete removal of the tumor results in the decreased value of the serodiagnosis if a detectable value can be observed in cancer-bearing state, and hence prognosis can be attained.

[0250] (3) Diagnosis in Cats

Next, cancer-bearing cats and healthy cats were diagnosed. Using the above-described partial polypeptide of canine CEP and anti-cat IgG antibody, the IgG 2 0 antibody titer of feline serum which specifically reacts with the polypeptide was measured in the same manner as described above. As a secondary antibody, HRPconjugated anti-cat IgG antibody (PEROXIDASE-CONJUGATED GOAT IgG FRACTION TO CAT IgG (WHOLE MOLECULE): manufactured by CAPPEL RESERCH REAGENTS) 8,000-fold diluted with the blocking solution was used.

[0251]

Feline Patient 1 (Chinchilla) underwent extirpation of mammary adenocarcinoma on August 17,2005. The absorbance at 450 nm was 0.48. In

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2017239516 04 Oct 2017

Feline Patient 2 (Himalayan), which underwent extirpation of ductal carcinoma on October 17,2006, the absorbance at 450 nm was 0.18. On the other hand, the absorbance was not detected in healthy cats at all.

[0252]

Thus, similarly to dogs, the absorbance value was detected in samples from cats suffering from cancer, while the absorbance value was not detected at all in samples from healthy cats. Hence, similarly to dogs, cancers in cats can also be detected by this method using a polypeptide derived from canine CEP.

[0253] (4) Diagnosis in Healthy Human

Using the above-described partial polypeptide of canine CEP and anti-human IgG antibody above, the IgG antibody titer of healthy human serum which specifically reacts with the polypeptide was measured in the same manner as described above. As a secondary antibody, HRP-conjugated anti-human IgG antibody (HRP-Goat Anti-Human IgG(H+L) Conjugate: manufactured by Zymed Laboratories) 10,000-fold diluted with the blocking solution was used. As a positive control, an immobilized ovalbumin antigen prepared by immobilizing 50 pg/ml ovalbumin in phosphate buffered saline on a solid phase was used. As a result, in Healthy Human 1, the absorbance at 450 nm observed on an ovalbumin

0 antigen was 0.25, while the absorbance at 450 nm observed on the recombinant protein was 0.02, hardly detected. Similarly, in Healthy Human 2, the absorbance at 450 nm observed on an ovalbumin antigen was 0.18, while the absorbance at 450 nm observed on the recombinant protein was 0.03, hardly detected.

[0254]

5 Further, the diagnosis was carried out in the same manner as described above using a full-length canine CEP having the sequence shown in SEQ ID NO:26 prepared in Example C-2. As a result, the diagnosis can be similarly attained in

125

2017239516 04 Oct 2017 humans, dogs and cats.

[0255]

Furthermore, the diagnosis was carried out in the same manner as described above using a full-length canine CEP having the sequence shown in SEQ ID NO:42 prepared in Example C-2. As a result, the diagnosis can be similarly attained in humans, dogs and cats.

[0256]

Example C-4: Cancer Diagnosis Using Polypeptide Derived from Human CEP Using the partial polypeptide of human CEP (SEQ ID NO.36; 1513rd to

2325th amino acid region of SEQ ID NO:28) prepared in Example C-2, the IgG antibody titer of human, canine and feline sera which react with the polypeptide was measured in the same manner as in Example C-3.

[0257]

The diagnosis was carried out using healthy human serum. In the same manner as in Example C-3 (4), ovalbumin antigen was used as a positive control.

As a result, the absorbance value was detected in the case where ovalbumin was immobilized on a solid phase, while the absorbance value was hardly detected in the case where the partial polypeptide of human CEP was immobilized on a solid phase. [0258]

0 Similarly, in healthy dogs and cats, the absorbance at 450 nm was hardly detected in the case where the polypeptide was immobilized on a solid phase.

[0259]

On the other hand, Canine Patient 11 (Shih Tzu) underwent extirpation of mammary adenocarcinoma on June 21,2007. According to the pathological

5 diagnosis using the extirpated tissue, the mammary gland tissue contained highly atypical, invasive ceils, and grew to form adenomatous hyperplasia showing large and small massive structures. Hence, this patient was diagnosed as malignant tumor.

126

2017239516 04 Oct 2017 in Canine Patient 11, the absorbance at 450 nm was 0.33. The malignancy diagnosis was carried out using additional 310 serum samples which had been diagnosed as malignant by pathological diagnosis. As a result, by diagnosing a sample showing twice the average value of healthy canine samples as malignant, 185 samples i.e. 59.5% of the malignant cases could be successfully diagnosed as malignant.

[0260]

Moreover, in Feline Patient 3 (Mixed Breed), which underwent extirpation of mammary adenocarcinoma on April 3,2007, the absorbance at 450 nm was 0.15.

[0261]

The above described results indicated that the diagnosis can also be similarly carried out in humans, dogs and cats by using a polypeptide derived from human CEP.

[0262]

Furthermore, pleural effusion and ascites samples collected from terminal cancer dogs were subjected to the diagnosis using the recombinant human protein in the same manner as the recombinant canine protein. As a result, values similar to those detected in serum samples could be detected, and hence cancer diagnosis could be successfully attained.

[0263]

In addition, the diagnosis was carried out in the same manner as described above using a full-length human CEP having the sequence shown in SEQ ID NO:28 prepared in Example C-2. As a result, the diagnosis could also be similarly carried out in humans, dogs and cats.

[0264]

Example C-5: Cancer Diagnosis by Measuring Antigen Polypeptide (1)

Mice and rabbits were immunized with the recombinant canine protein having

127

2017239516 04 Oct 2017 the sequence shown in SEQ ID NO :3 5 prepared in Example C-2 to obtain an antibody specific to this antigen. Using this polyclonal antibody, detection of the antigen polypeptide per se contained in the serum from cancer bearing living body was carried out by sandwich ELISA. Using anti-mouse IgG antibody, the amount of 5 the protein in the serum which specifically reacts with the prepared polyclonal antibody specific to the protein was measured by sandwich ELISA.

[0265]

As for immobilization of a primary antibody on a solid phase, 100 μΕ/well of the rabbit antiserum 20-fold diluted with phosphate buffered saline was added to a 10 96-well Immobilizer Amino plate (manufactured by Nunc), and the plate was shaken at room temperature for 2 hours. As for blocking, 100 μL/well of 50 mM sodium bicarbonate buffer (pH 8.3) containing 0.5% BSA (bovine serum albumin, manufactured by Sigma Aldrich Japan) (hereinafter referred to as blocking solution) was added to the plate, and the plate was shaken at room temperature for 1 hour. To 15 the plate, 100 pL/well of the serum from cancer-bearing body diluted with the blocking solution was added, and the plate was shaken at room temperature for 3 hours to allow the reaction to proceed. As for the diluted serum, a 10-fold serial dilution ranging 10 to 1,000-fold was prepared. After washing the wells 3 times with phosphate buffered saline containing 0.05% Tween20 (manufactured by Wako 2 0 Pure Chemicals) (hereinafter referred to as PBS-T), 100 pL/well of mouse antiserum 200-fold diluted with the blocking solution was added thereto, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 pL/well of HRP-conjugated mouse IgG antibody (Stabilized Goat Anti Mouse HRP conjugated : manufactured by PIERCE) 2 5 2,000-fold diluted with the blocking solution was added thereto as a tertiary antibody, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 μΐ/well of a HRP

128

2017239516 04 Oct 2017 substrate, TMB (1-Step Turbo TMB (tetramethylbenzidine), manufactured by PIERCE), was added thereto, and the enzyme-substrate reaction was allowed to proceed at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding 100 μΙ/well of 0.5 M sulfuric acid solution (manufactured by Sigma

Aldrich Japan), and then the absorbance was measured at 450 nm with a microplate reader. As a control, a plate on which the rabbit antiserum was not immobilized and a plate with which serum from a cancer-bearing body was not reacted were measured in the same manner as described above.

[0266]

Asa result, the polypeptide was detected in cancer-bearing dogs and cats suffering from cutaneous leiomyosarcoma, breast cancer, malignant melanoma or the like, while the polypeptide was not detected in healthy dogs, healthy cats and healthy humans. Hence, cancers could also be diagnosed by this method in which the antigen polypeptide was detected with an antibody which was prepared by using the recombinant canine polypeptide as an immunogen.

[0267]

In addition, the diagnosis was carried out in the same manner as described above using an antibody which was prepared by using as an immunogen the fulllength canine CEP having the sequence shown in SEQ ID NO:26 prepared in

0 Example C-2.

[0268]

As a result, cancers could also be diagnosed in dogs and cats by this method in which the antigen polypeptide was detected with an antibody which was prepared by using a full-length canine CEP as an immunogen.

[0269]

Furthermore, the diagnosis was earned out in the same manner as described above using an antibody which was prepared by using as an immunogen the full129

2017239516 04 Oct 2017 length canine CEP having the sequence shown in SEQ ID NO :42 prepared in Example C-2.

[0270]

As a result, cancers could also be diagnosed in dogs and cats by this method in which the antigen polypeptide was detected with an antibody which was prepared by using a full-length canine CEP as an immunogen.

[0271]

Example C-6: Cancer Diagnosis by Measuring Antigen Polypeptide (2)

Mice and rabbits were immunized with the recombinant human protein having the sequence shown in SEQ ID NO :3 6 prepared in Example C-2 to obtain an antibody specific to this antigen. In the same manner as in Example C-5, detection of the antigen polypeptide per se contained in the serum from cancer-bearing living body was carried out by sandwich ELISA using this polyclonal antibody.

[0272]

Asa result, the polypeptide was detected in cancer-bearing dogs and cats suffering from cutaneous leiomyosarcoma, breast cancer, malignant melanoma or the like, while the polypeptide was not detected in healthy dogs, healthy cats and healthy humans. Hence, cancers could also be diagnosed by this method in which the antigen polypeptide was detected with an antibody prepared by using the recombinant

0 human polypeptide as an immunogen.

[0273]

In addition, the diagnosis was carried out in the same manner as described above using an antibody which was prepared by using as an immunogen the fulllength human CEP having the sequence shown in SEQ ID NO:28 prepared in

Example C-2.

[0274]

As a result, cancers could also be diagnosed in dogs and cats by this method

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2017239516 04 Oct 2017 in which the antigen polypeptide was detected with an antibody which was prepared by using a full-length human CEP as an immunogen.

[0275]

Example D-l: Acquisition of Novel Cancer Antigen Protein by SEREX Method (1) Preparation of cDNA Library

Total RNA was prepared from testis tissue of a healthy dog by the Acid guanidium-Phenol-Chloroform method, and poly(A) RNA was purified using Oligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co., Ltd.) in accordance with the protocol attached to the kit.

[0276]

Using the obtained mRNA (5 pg), a dog testis cDNA phage library was synthesized. Preparation of the cDNA phage library was carried out using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit (manufactured by STRATAGENE) in accordance with the protocols attached to 15 the kits. The size of the prepared cDNA phage library was 1.3 ^x 10⁶ pfu/ml.

[0277] (2) Screening of cDNA Library with Serum

Using the dog testis-derived cDNA phage library prepared as described above, immunoscreening was carried out. More particularly, host E. coli cells (XL 1-Blue 2 0 MRF') were infected with the library such that 2,340 clones should appear on an

NZY agarose plate having the size of Φ90 x 15 mm, and cultured at 42°C for 3 to 4 hours to allow the phage to form plaques. The plate was covered with nitrocellulose membrane (Hybond C Extra: manufactured by GE Healthcare Bio-Science) impregnated with IPTG (isopropyl-β-D-thiogalactoside) at 37°C for 4 hours to induce 2 5 and express proteins, which were thus transferred to the membrane. Subsequently, the membrane was recovered and soaked in TBS (10 mM Tris-HCl, 150 mM NaCl; pH 7.5) containing 0.5% non-fat dry milk, followed by shaking at 4°C overnight to

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2017239516 04 Oct 2017 suppress non-specific reactions. This filter was allowed to react with 500-fold diluted canine patient serum at room temperature for 2 to 3 hours.

[0278]

As the above-described canine patient serum, serum collected from canine patients suffering from breast cancer was used. The serum was stored at -80°C and pretreated immediately before use. The method of the pretreatment of the serum was as follows. That is, host E. coli cells (XL 1-Blue MRF') were infected with λ ZAP Express phage to which no foreign gene was inserted, and then cultured on NZY plate medium at 37°C overnight. Subsequently, the buffer of 0.2 M NaHCCh, 10 pH 8.3 containing 0.5 M NaCl was added to the plate, and the plate was left to stand at 4°C for 15 hours, followed by collecting the supernatant as an E. coli/phage extract. Thereafter, the collected E. co/z/phage extract was allowed to flow through an NHS column (manufactured by GE Healthcare Bio-Science) to immobilize proteins derived from the E. coli/phage. thereon. The serum from the canine patients was allowed to flow through and react with this protein-immobilized column to remove antibodies adsorbed on E. coli and/or the phage. The serum fraction that passed through the column was 500-fold diluted with TBS containing 0.5% non-fat diy milk, and the resulting diluent was used as the material for the immunoscreening.

[0279]

0 The membrane on which the thus treated serum and the above-described fusion protein were blotted was washed 4 times with TBS-T (0.05% Tween 20/TBS), and allowed to react with goat anti-dog IgG (Goat anti Dog IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5,000-fold diluted with TBS containing 0.5% non-fat dry milk as a secondary antibody at room temperature for 1 hour,

5 followed by detection by the enzyme coloring reaction using the NBT/BCIP reaction solution (manufactured by Roche). Colonies at positions where a positive coloring reaction was observed were recovered from the NZY agarose plate having the size of

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2017239516 04 Oct 2017

Φ90 x 15 mm, and dissolved in 500 μΐ of SM buffer (100 mM NaCl, 10 mM MgClSO4, 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). The screening was repeated as a second and third screening in the same manner as described above until a single coloring reaction-positive colony was obtained, thereby isolating one positive clone after screening of 30,940 phage clones reactive with IgG in the serum.

[0280] (3) Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-described method to a base sequence analysis, an operation of conversion of the phage vector to a plasmid 10 vector was carried out. More particularly, 200 μΐ of a solution prepared to contain a host E. coli (XLl-Blue MRF) such that the absorbance ODgoo should be 1.0 was mixed with 100 μΐ of a purified phage solution and further with 1 μΐ of ExAssist helper phage (manufactured by STRATAGENE), and the reaction was allowed to proceed at 37°C for 15 minutes. To the reaction mixture, 3 ml of LB medium was added, and the mixture was cultured at 37°C for 2.5 to 3 hours, followed by immediate incubation in a water bath at 70°C for 20 minutes. The mixture was then centrifuged at 4°C at 1,000 xg for 15 minutes, and the supernatant was recovered as a phagemid solution. Subsequently, 200 μΐ of a solution prepared to contain a phagemid host E. coli (SOLR) such that the absorbance OD^oo should be 1.0 was

0 mixed with 10 μΐ of a purified phage solution, and the reaction was allowed to proceed at 37°C for 15 minutes. Thereafter, 50 μΐ of the reaction mixture was plated on ampicillin (final concentration: 50 pg/ml)-containing LB agar medium, and cultured at 37°C overnight. A single colony of transformed SOLR was recovered and cultured in ampicillin (final concentration: 50 pg/ml)-containing LB medium at

5 37°C, followed by purification of plasmid DNA having an insert of interest using

QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

[0281]

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The purified plasmid was subjected to an analysis of the entire sequence of the insert by the primer walking method using the T3 primer described in SEQ ID NO:5 and the T7 primer described in SEQ ID NO:6. By this sequence analysis, the gene sequence described in SEQ ID NO:44 was obtained. Using the base sequence and the amino acid sequence of this gene, homology search against known genes was carried out using a homology search program BLAST (http://www.ncbi.nIm.nih.gov/BLAST/). As a result, it was revealed that the obtained gene is the TRIP 11 gene. The human homologous factor of canine TRIP 11 was human TRIP11 (homology: base sequence, 88%; amino acid sequence,

86%). The base sequence of human TRIP11 is shown in SEQ ID NO :46, and the amino acid sequence thereof is shown in SEQ ID NO:47.

[0282] (4) Analysis of Expression in Each Tissue

The expression of the gene, which was obtained by the above-described method, in normal tissues and various cell lines of dog and human were investigated by the RT-PCR (Reverse Transcription-PCR) method. The reverse transcription reaction was carried out as follows. That is, total RNA was extracted from 50 to 100 mg of each tissue or 5 to 10 * 10⁶ cells of each cell line using TRIZOL reagent (manufactured by Invitrogen) in accordance with the protocol attached to the kit.

0 Using this total RNA, cDNA was synthesized by Superscript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen) in accordance with the protocol attached to the kit. As the cDNAs from human normal tissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA (manufactured by Invitrogen), QUICKClone cDNA (manufactured by CLONTECH) and Large-Insert cDNA Library

5 (manufactured by CLONTECH) were used. The PCR reactions were carried out as follows using primers (described in SEQ ID NOs:48 and 49) specific to the obtained gene. That is, respective reagents and the attached buffer were mixed such that the

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2017239516 04 Oct 2017 mixture should contain 0.25 μΐ of the sample prepared by the reverse transcription reaction, 2 μΜ each of the above primers, 0.2 mM each of dNTP and 0.65 U of ExTaq polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 25 μΐ, and the reaction was carried out with 30 cycles of 94°C for 30 seconds, 55°C for

30 seconds and 72°C for 1.5 minutes using Thermal Cycler (manufactured by BIO

RAD). The above-described gene-specific primers were those which amplify the regions of the 1519th to 2957th bases of the base sequence of SEQ ID NO:44 (canine TRIP 11 gene) and the 1872nd to 3310th bases of the base sequence of SEQ ID NO :46 (human TRIP 11 gene), and can be used for investigation of the expression of both the canine TRIP 11 gene and the human TRIP 11 gene. As a control for comparison, primers (described in SEQ ID NOs:9 and 10) specific to GAPDH were used simultaneously. As a result, as shown in Fig. 13, strong expression of the canine TRIP 11 gene was observed in testis among the normal dog tissues, and on the other hand, strong expression was observed in the canine breast cancer cell line.

Expression of the human TRIP 11 gene was confirmed, as is the case with the canine

TRIP 11 gene, only in testis among the human normal tissues, but the expression was detected in many types of cancer cell lines such as brain tumor, leukemia, breast cancer, lung cancer and esophagus cancer cell lines among human cancer cell lines. Thus, the human TRIP 11 gene was also confirmed to be specifically expressed in

0 testis and cancer cells.

[0283]

In Fig. 13, reference numeral 1 in the ordinate indicates the expression pattern of the TRIP 11 gene, and reference numeral 2 indicates the expression pattern of the GAPDH gene as a control for comparison.

[0284]

Example D-2: Preparation of Canine and Human TRIP11 proteins (1) Preparation of Recombinant Protein

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2017239516 04 Oct 2017

Based on the gene of SEQ ID NO:44 obtained in Example D-l, a recombinant protein was prepared by the following method. Respective reagents and the attached buffer were mixed such that the mixture should contain 1 pl of the vector which was prepared from the phagemid solution obtained in Example D-l and was 5 subjected to the sequence analysis, 0.4 pM each of two kinds of primers having Sall and ATioI restriction sites (described in SEQ ID NOs:50 and 51), 0.2 mM dNTP and 1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 50 pl, and PCR was carried out with 30 cycles of 98°C for 10 seconds, 55°C for 5 seconds and 72°C for 6 minutes using Thermal Cycler (manufactured by BIO RAD). Using the above-described two kinds of primers, a region encoding an amino acid region (SEQ ID NO:54) of 237th to 1023rd amino acids of SEQ ID NO:45 is obtained. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 2.4 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0285]

In the same manner, PCR was carried out using two kinds of primers described in SEQ ID NOs:56 and 57 to obtain a region encoding the entire amino acid sequence of SEQ ID NO:45. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 6.0 kbp was

0 purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0286]

Each of the purified DNA fragments was ligated into a cloning vector pCRBlunt (manufactured by Invitrogen). E. coli was transformed with the resulting ligation product, and plasmids were recovered thereafter, followed by confirming, by 2 5 sequencing, that the amplified gene fragment matches the sequence of interest. The plasmid that matched the sequence of interest was treated with restriction enzymes Sail and Xhol and purified using QIAquick Gel Extraction Kit, followed by inserting

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2017239516 04 Oct 2017 the gene sequence of interest into an expression vector for E. coli, pET30b (manufactured by Novagen) that had been treated with Salt and XhoL Usage of this vector enables production of a His-tag fusion recombinant protein. E. coli for expression, BL21 (DE3), was transformed with this plasmid, and expression of the protein of interest was induced in E: coli with 1 mM IPTG.

[0287]

Further, based on the gene of SEQ ID NO:46, a recombinant protein of the human homologous gene was prepared by the following method. Respective reagents and the attached buffer were mixed such that the mixture should contain 1 10 μΐ of the cDNA prepared in Example D-l whose expression could be confirmed by the RT-PCR method in various tissues/cells, 0.4 μΜ each of two kinds of primers having Ndel and KpriL restriction sites (described in SEQ ID NOs:52 and 53), 0.2 mM dNTP and 1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of 50 μΐ, and PCR was carried out with 30 cycles 15 of 98°C for 10 seconds, 55°C for 5 seconds and 72°C for 6 minutes using Thermal Cycler (manufactured by BIO RAD). Using the above-described two kinds of primers, a region encoding an amino acid region (SEQ ID NO:55) of 236th to 1023rd amino acids of SEQ ID NO:47 is obtained. After the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 2.4 2 0 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0288]

In the same manner, PCR was carried out using two kinds of primers described in SEQ ID NOs:58 and 59 to obtain a region encoding the entire amino acid sequence of SEQ ID NO:47. After the PCR, the amplified DNA was subjected 25 to electrophoresis using 1% agarose gel, and a DNA fragment of about 6.0 kbp was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

[0289]

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Each of the purified DNA fragments was ligated into a cloning vector pCRBlunt (manufactured by Invitrogen). E. coli was transformed with the resulting ligation product, and plasmids were recovered thereafter, followed by confirming, by sequencing, that the amplified gene fragment matches the sequence of interest. The plasmid that matched the sequence of interest was treated with restriction enzymes

Ndel and Kpnl and purified using QIAquick Gel Extraction Kit, followed by inserting the gene sequence of interest into an expression vector for E. coli, pET30b (manufactured by Novagen) that had been treated with Ndel and Kpnl. Usage of this vector enables production of a His-tag fusion recombinant protein. E. coli for expression, BL21 (DE3), was transformed with this plasmid, and expression of the protein of interest was induced in E. coli with 1 mM IPTG.

[0290] (2) Purification of Recombinant Proteins

The above-obtained recombinant E. coli cells that expressed a part of SEQ ID

NO:44 and a part of SEQ ID NO:46, respectively, were cultured in kanamycin (final concentration: 30 pg/ml)-containing LB medium at 37°C until the absorbance at 600 nm reached about 0.7, and then IPTG was added thereto such that its final concentration should be 1 mM, followed by culturing them at 30°C for 20 hours. Subsequently, the cells were collected by centrifugation at 4,800 rpm for 10 minutes.

0 The pellet of the cells was suspended in phosphate-buffered saline and further subjected to centrifugation at 4,800 rpm for 10 minutes to wash the cells. [0291]

The cells were suspended in phosphate-buffered saline and subjected to sonication on ice. The sonicated solution of E. coli was centrifuged at 7,000 rpm 25 for 15 minutes to obtain the supernatant as the soluble fraction and the precipitate as the insoluble fraction.

[0292]

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2017239516 04 Oct 2017

The insoluble fraction was suspended in 4% Triton X-100 solution and the resulting suspension was centrifuged at 7,000 rpm for 10 minutes. This operation was repeated twice and an operation of removal of proteases was carried out. Thereafter, the residue was suspended in phosphate-buffered saline and an operation 5 of removal of the surfactant was carried out.

[0293]

The residue was suspended in 6M guanidine hydrochloride (manufactured by Sigma Aldrich Japan)-containing 20 mM phosphate buffer (pH 8.0), and the resulting suspension was left to stand at 4°C for 15 hours to denature proteins. Thereafter, the suspension was centrifuged at 7,000 rpm for 20 minutes, and the obtained soluble fraction was placed in a nickel chelate column prepared by a conventional method (carrier: Chelating Sepharose (trademark) Fast Flow (GE Health Care); column volume: 5mL; equilibration buffer: 6M guanidine hydrochloride-containing 20 mM phosphate buffer (pH 8.0)). The fraction that was not adsorbed to the column was washed away with 10 column volumes of 6 M sodium chloride-containing 20 mM phosphate buffer (pH 8.0) and 10 mM imidazole-containing 20 mM phosphate buffer (pH 8.0), and elution was immediately carried out with a four-step density gradient of 50 mM-500 mM imidazole. Five column volumes of the eluted fractions was collected in each elution step. Elution of the proteins of interest was confirmed by

0 Coomassie staining carried out according to a conventional method. Based on the result, the eluted fractions were desalted and concentrated to obtain the material to be solid-phased for diagnosis.

[0294]

In the same maimer, the recombinant E. coli cells that expressed the full2 5 length of SEQ ID NOs:45 and 47, respectively, were cultured, and the proteins of interest were purified to obtain the material to be solid-phased for diagnosis. [0295]

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Example D-3: Cancer Diagnosis Using Polypeptide Derived from Canine TRIP 11 (1) Cancer Diagnosis in Dogs

Blood samples were collected from 486 canine patients in which malignant or benign tumors were found and 6 healthy dogs, and sera were separated therefrom.

Using the partial polypeptide of canine TRIP 11 (SEQ ID NO:54; 237th to 1023rd amino acid region of SEQ ID NO:45) prepared in Example D-2 and anti-dog IgG antibody, the IgG antibody titer of the sera which specifically react with the polypeptide was measured by ELISA.

[0296]

As for immobilization of the prepared protein on a solid phase, 100 pL/well of a solution of the recombinant protein diluted to 50 pg/mL with phosphate buffered saline was added to a 96-well Immobilizer Amino plate (manufactured by Nunc), and the plate was left to stand at 4°C overnight. As for blocking, 100 pL/well of 50 mM sodium bicarbonate buffer (pH 8.3) containing 0.5% BSA (bovine serum albumin, manufactured by Sigma Aldrich Japan) (hereinafter referred to as blocking solution) was added to the plate, and the plate was shaken at room temperature for 1 hour. Serum sample was 1,000-fold diluted with the blocking solution, and 100 pL/well of the diluted serum was added to the plate, followed by shaking the plate at room temperature for 3 hours to allow the reaction to proceed. After washing the wells 3

0 times with phosphate buffered saline containing 0.05% Tween20 (manufactured by Wako Pure Chemicals) (hereinafter referred to as PBS-T), 100 pL/well of HRPconjugated dog IgG antibody (Goat anti Dog IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 3,000-fold diluted with the blocking solution was added thereto, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 μΐ/well of a HRP substrate, TMB (1-Step Turbo TMB (tetramethylbenzidine), manufactured by PIERCE), was added thereto, and the enzyme-substrate reaction was allowed to

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2017239516 04 Oct 2017 proceed at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding 100 μΐ/well of 0.5 M sulfuric acid solution (manufactured by Sigma Aldrich Japan), and then the absorbance was measured at 450 nm with a microplate reader. As a control, a plate on which the prepared recombinant protein was not immobilized and a plate with which the serum from a cancer-bearing dog was not reacted were measured in the same manner as above.

[0297]

Among the total 486 samples used in the above-described cancer diagnosis,

311 samples were definitely diagnosed as malignant by pathological diagnosis using the extirpated tumor tissue.

[0298]

Specifically, the samples were diagnosed as cancer such as malignant melanoma; malignant mixed tumor; hepatocellular carcinoma; basal cell carcinoma; acanthomatous epulis; intraoral tumor; perianal adenocarcinoma; anal sac tumor; anal 15 sac apocrine carcinoma; Sertoli cell tumor; vulva cancer; sebaceous adenocarcinoma; sebaceous epithelioma; sebaceous adenoma; sweat gland carcinoma; intranasal adenocarcinoma; nasal adenocarcinoma; thyroid cancer; colon cancer; bronchial adenocarcinoma; adenocarcinoma; ductal carcinoma; mammary adenocarcinoma; combined mammary adenocarcinoma; mammary gland malignant mixed tumor;

0 intraductal papillary adenocarcinoma; fibrosarcoma; hemangiopericytoma; osteosarcoma; chondrosarcoma; soft tissue sarcoma; histiocytic sarcoma; myxosarcoma; undifferentiated sarcoma; lung cancer; mastocytoma; cutaneous leiomyoma; intra-abdominal leiomyoma; leiomyoma; squamous cell carcinoma; chronic lymphocytic leukemia; lymphoma; gastrointestinal lymphoma; digestive

5 organ lymphoma; small cell or medium cell lymphoma; adrenomedullary tumor; granulosa cell tumor; pheochromocytoma; bladder cancer (transitional cell carcinoma); suppurative inflammation; intra-abdominal liver tumor; liver cancer;

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2017239516 04 Oct 2017 plasmacytoma; malignant hemangiopericytoma; angiosarcoma; anal sac adenocarcinoma; oral cancer; metastatic malignant melanoma; amelanotic malignant melanoma; cutaneous malignant melanoma; malignant myoepithelioma; malignant seminoma; seminoma; adenocarcinoma of the large intestine; gastric adenocarcinoma; low-grade sebaceous carcinoma; ceruminous adenocarcinoma; apocrine carcinoma; poorly differentiated apocrine sweat gland carcinoma; malignant fibrous histiocytoma; multiple myeloma; mesenchymal malignant tumor; Iiposarcoma; osteosarcoma; sarcoma of unknown origin; soft part sarcoma (spindle cell tumor); poorly differentiated sarcoma; synovial sarcoma; angiosarcoma;

metastatic malignant epithelioma; tubular mammary adenocarcinoma; mammary ductal carcinoma; inflammatory breast cancer; germinoma; leukemia; invasive trichoepithelioma; medium cell lymphoma; multicentric lymphoma; osteosarcoma (mammary gland); mastocytoma (Patnaik II type); mastocytoma (Grade II); leiomyosarcoma or the like.

[0299]

As shown in Fig. 15, sera from these cancer-bearing dogs showed a significantly high antibody titer against the recombinant protein. It was revealed that, by diagnosing a sample showing twice the average value of healthy canine samples as malignant, 78 samples i.e. 25.1% of the malignant cases could be successfully diagnosed as malignant. The details of these 78 cancer samples are as follows. It is noted that the following number of each cancer case is a cumulative total, as some samples contained multiple primaries.

Malignant melanoma, 4 cases; lymphoma, 5 cases; suppurative inflammation, 1 case; granulosa cell tumor, 1 case; hepatocellular carcinoma, 2 cases; malignant testicular 25 tumor, 2 cases; intraoral tumor, 3 cases; perianal adenoma, 5 cases; osteosarcoma, 2 cases; ductal carcinoma, 6 cases; mammary adenocarcinoma, 16 cases; combined mammary adenocarcinoma, 8 cases; lung cancer, 1 case; sebaceous adenocarcinoma,

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2017239516 04 Oct 2017 cases; mastocytoma, 6 cases; leiomyosarcoma, 2 cases; squamous cell carcinoma, 4 cases; malignant mixed tumor, 1 case; metastatic malignant melanoma, 1 case; mammary ductal carcinoma, 1 case; apocrine carcinoma, 1 case; gastric adenocarcinoma, 1 case; multicentric lymphoma, 1 case; seminoma, 1 case;

plasmacytoma, 1 case.

[0300]

The above-described diagnostic method was also carried out using pleural effusion samples and ascites samples collected from terminal cancer dogs. As a result, values similar to those detected in serum samples could be detected, and hence 10 cancer diagnosis could be successfully attained.

[0301]

Furthermore, it was confirmed that diagnostic approaches such as diagnosis of cancers existing in an invisible part of the body, assessment of cancer stage and grade, follow-up of postoperative patients, diagnosis of recurrence and metastasis and the 15 like can also be attained by applying the above-described diagnostic method. The followings are several of the practical examples of the detailed diagnosis shown in Fig. 4.

[0302] (2)-1 Diagnosis of Invisible Tumors

In Canine Patient 1 (Flat-Coated Retriever), any tumors were not found on

June 7, 2007. But about 20 days later, on June 24,2007, a pedunculated tumor with a diameter of 2 mm was found on the gum at the root of the canine tooth. The tumor was ligated at its pedunculated part and excised on the day it found. The absorbance at 450 nm observed before the tumor became visible with the naked eye

5 was 0.15, which was significantly high and not so different from the absorbance at the time of finding tumor, 0.14. The result indicates that it is possible to diagnose cancers even in an invisible part such as an intraperitoneal part by the method of the

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2017239516 04 Oct 2017 present invention.

[0303]

Rise of the value was observed before the tumor became visible with the naked eye, which is considered to have been a sign of tumor development. Thus, the method of the present invention is useful in medical examinations such as periodic health examination.

[0304]

Canine Patient 1 was again checked by the serodiagnosis 2 weeks after the tumor excision. As a result, the absorbance at 450 nm was 0, not detected. Thus, 10 it was also confirmed that the cancer antigen-expressing tumor which had caused the increased antibody titer was completely removed (see, (2)-4, Follow-Up of Postoperative Patients).

[0305] (2)-2 Assessment of Stage of Cancer Progression

The stage of cancer progression is determined based on the size or depth of the tumor, how much the tumor exerts influence on the surrounding tissues, whether the tumor metastasizes or not, and the like. It was revealed herein that the detected value is higher than before if the metastasis occurs, i.e., the cancer has advanced. [0306]

0 (2)-3 Assessment of Grade of Cancer Malignancy

Basaliomas include malignant type and benign type. Recently, according to the new WHO classification, malignant basaliomas are called basal cell carcinoma, and benign basaliomas are called trichoblastoma.

[0307]

5 Canine Patient 2 (Beagle) was diagnosed as basal cell carcinoma (malignant).

The serodiagnosis was carried out at the time of the surgery. As a result, the absorbance at 450 nm was 0.15. On the other hand, in Canine Patient 3 (Mixed

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2017239516 04 Oct 2017

Breed) diagnosed as trichoblastoma (benign), the serodiagnosis carried out at the time of the surgery revealed that the absorbance at 450 nm was 0, not detected at all. Thus, even in the case of the same basaliomas, malignant basal cell carcinoma and benign trichoblastoma can be distinctively diagnosed.

[0308]

Next example is mammary gland tumors. Mammary gland tumors include malignant tumors such as mammary adenocarcinoma and mammary gland malignant mixed tumor, and benign mammary tumors which do not show malignant symptoms. Canine Patient 4 (Yorkie) underwent extirpation of mammary gland malignant mixed 10 tumor and mammary adenocarcinoma on May 17,2006. In general, the complete excision of mixed tumors in mammary gland is easy because they are poorly invasive to the surrounding tissues even if they are malignant, and thus the postoperative course of the patients is usually uneventful. However, Canine Patient 4 had been diagnosed as highly malignant tumor, because the pathological diagnosis using the extirpated tissue revealed that some components of the specimen from Canine Patient 4 showed an invasive nature. On the other hand, mammary adenocarcinoma is a highly invasive tumor which often recurs and metastasizes. Although invasion of the tumor cells was not observed in the specimen from Canine Patient 4, it had been pointed out that highly malignant components possibly proliferated in other region

0 out of the specimen. Thus, the findings in the pathological diagnosis clearly taught that Canine Patient 4 was suffering from highly malignant mammary cancer. A blood sample was collected during the surgery and the serodiagnosis was carried out to find that the absorbance at 450 nm was 0.20. Canine Patient 5 (Yorkshire Terrier) underwent extirpation of mammary tumor on January 28,2007. According to the pathological diagnosis using the extirpated tissue, atypism of cells was low, and thus Canine Patient 5 was diagnoses as benign mastadenoma without malignant findings. A blood sample was collected during the surgery and the serodiagnosis

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2017239516 04 Oct 2017 was carried out to find that the absorbance at 450 nm was 0. The results in the two cases above revealed that highly malignant tumors show a higher value than low malignant, benign tumors.

[0309] (2)-4 Follow-Up of Postoperative Patients

Canine Patient 6 (Shih Tzu) visited the hospital due to an intraoral tumor and underwent the extirpation on March 22,2007. The serodiagnosis was carried out at that time. As a result, the absorbance at 450 nm was 0.12. In addition, based on the pathological diagnosis using the extirpated tissue, Canine Patient 6 was diagnosed as malignant acanthomatous epulis. This kind of tumor often recurs if excision is insufficient, though distant metastasis seldom happens. Thus, it is important whether the tumor can be completely excised by surgery or not. According to the follow up on May 18,2007, the absorbance at 450 nm was 0.02 and hence the antibody titer was decreased. Recurrence has not been found till August of 2007. Thus, it is considered that the value detected by the serodiagnosis became lower than that obtained at the time of surgery because the tumor could be completely excised from Canine Patient 6.

[0310]

Canine Patient 7 (Yorkie) was diagnosed by the serodiagnosis using a serum

0 sample collected on May 17,2006, and the absorbance at 450 nm was 0.20. This patient visited the hospital on December 16,2006 for follow-up, and the serodiagnosis was carried out again. As a result, the absorbance at 450 nm was 0. No recurrence nor metastasis has been found till August of 2007. Thus, it is considered that the value detected by the serodiagnosis became lower than that

5 obtained at the time of surgery because the tumor could be completely excised from Canine Patient 7.

[0311]

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2017239516 04 Oct 2017 (2)-5 Diagnosis of Recurrence

Canine Patient 8 (Husky) underwent an extirpation of mammary adenocarcinoma on May 8,2007. The serodiagnosis was carried out at the time of the surgery, and the absorbance at 450 nm was 0,04. The pathological diagnosis using the extirpated tissue revealed that highly atypical epithelial cells proliferated and mainly formed ductal structures, and thus this patient was diagnosed as primary breast adenocarcinoma. It was said that the patient was at a high risk of recurrence or metastasis to lymph nodes or distant organs, as many cancer cells had already entered the lymph vessels at that time. On June 28,2007, about one and a half months after the surgery, metastasis was found at the same site. The serodiagnosis was carried out at that time to find that the value increased to 0.07. Thus, it was confirmed that the value detected by the serodiagnosis was higher in the end of June than in the beginning of May because the tumor could not have been completely excised or recurrence would have occurred in Canine Patient 8.

[0312] (2)-6 Diagnosis of Metastasis

Canine Patient 9 (Scottish Terrier), repeatedly undergoing metastasis and recurrence, was diagnosed as mammary tumor in February of 2003; intraoral malignant melanoma in August of 2003; malignant melanoma of the lip in January of 2 0 2005; and as intraoral melanoma on April 13,2005, all of which were excised by surgery. This patient visited the hospital again on December 17,2006 for follow-up after the recurrence of intraoral melanoma in April of 2005, and the serodiagnosis was carried out at that time to find that the absorbance at 450 nm was 0. Half a year later, on June 20,2007, the patient again visited the hospital because of hypertrophy 25 of cervical and malar lymph nodes. In the case of lymphomas, hypertrophy of lymph nodes is systemically observed. Because Canine Patient 9 had only two swollen lymph nodes, this patient was clinically diagnosed as probable metastatic

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2017239516 04 Oct 2017 lymphoma. The diagnosis according to the present invention also revealed that it was a tumor which had metastasized from the tumor previously existed in this patient as the absorbance at 450 nm greatly increased to 0.27.

[0313]

Canine Patient 10 (Shiba Inu) underwent extirpation of oral malignant melanoma of the right lip on March 11,2006. This patient has a history of anticancer drug treatment (cyclophosphamide) from June 10 to September 26 in 2006, and has received BIREMO S, which contains organic germanium as a main ingredient, since May 23, 2006. On March 20,2007, this patient underwent extirpation of a tumor which was considered to be metastasis from the tumor mentioned above, and the serodiagnosis was carried out. As a result, the absorbance at 450 nm was approximately 0, hardly detected. Based on the pathological diagnosis using the tissue extirpated at that time, Canine Patient 10 was diagnosed as metastatic malignant melanoma. On June 27,2007, three months after the extirpation of metastatic melanoma, metastasis occurred in this patient again. The tumor which was extirpated on March 20,2007 existed in the right cervical part, and the tumor which occurred on June 27, 2007 was on the opposite side. As for the shape of the tumor, a black mass was formed similarly to the previous tumor. The tumor, having the size of 3.1 ><3.2 x 0.8 cm, was also clinically diagnosed as

0 metastasis. The serodiagnosis was carried out again to find that the absorbance at

450 nm increased to 0.02, which indicated that it was metastatic tumor from the previous one.

[0314] (2)-7 Therapy Monitoring

5 Canine Patient 12 (Miniature Dachshund) underwent tumor extirpation on

April 19,2007. According to the pathological diagnosis using the extirpated tumor, the patient was suffering from moderately-malignant combined mammary

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2017239516 04 Oct 2017 adenocarcinoma with a high probability of invasive and metastatic development. The serodiagnosis was carried out at that time, and the absorbance at 450 nm was 0.03. On June 3,2008, about one year after the extirpation, the serodiagnosis was carried out to find that the absorbance at 450 nm was 0, not detected at all.

Although any recurrent tumors were not found with the naked eye, an anticancer drug (INTERCAT) was administered once-weekly for 2 months to prevent recurrence. The serodiagnosis was carried out 2,4, and 6 weeks after the administration of the anticancer drug started. As a result, the absorbance at 450 nm was 0, not detected at all, in all trials. These results obtained in Canine Patient 12 confirmed that the value becomes lower than that detected in a cancer-bearing state if tumors can be completely removed, as well as that the value does not increase if anticancer drug treatment successfully prevents cancer metastasis, and thus change in treated patients can be followed. In addition, the diagnosis of recurrence can also be carried out as shown by Canine Patient 8, which confirms that the therapy monitoring can also be made possible.

[0315] (2)-8 Diagnosis of Malignancy of Recurrent Tumor

Canine Patient 13 (Golden Retriever) underwent tumor extirpation on May 1, 2005. The pathological diagnosis using the extirpated tumor revealed that the tumor 20 in this patient was malignant neoplastic lesion originated from mammary ductal epithelium, i.e., malignant mammary ductal carcinoma and malignant papillary carcinoma continuously growing through the mammary ducts. On June 28, 2008, about 3 years thereafter, tumor was found again and thus extirpation was carried out. The pathological diagnosis using the extirpated tumor revealed that nothing but

5 severe infiltration of inflammatory cells such as neutrophils, macrophages, plasma cells and the like could be observed around surgical sutures under the skin which was considered to be the previous surgical wound, and thus the patient was diagnosed as

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2017239516 04 Oct 2017 having no neoplastic lesions. According to the serodiagnosis carried out at that time, the absorbance at 450 nm was 0, not detected at all. The results observed in Canine Patients 8 and 13 indicated that the value of the serodiagnosis does not decrease or is sustained in cases where the recurrent tumor is malignant, and is not detected in cases 5 where the tumor is benign.

[0316] (3) Diagnosis in Cats

Next, cancer-bearing cats and healthy cats were diagnosed. Using the above-described partial polypeptide of canine TRIP11 and anti-cat IgG antibody, the

IgG antibody titer of feline serum which specifically reacts with the polypeptide was measured in the same manner as described above. As a secondary antibody, HRPconjugated anti-cat IgG antibody (PEROXIDASE-CONJUGATED GOAT IgG FRACTION TO CAT IgG (WHOLE MOLECULE): manufactured by CAPPEL RESERCH REAGENTS) 8,000-fold diluted with the blocking solution was used.

[0317]

Feline Patient 1 (Chinchilla) underwent tumor extirpation of mammary adenocarcinoma on August 17,2005. The absorbance at 450 iim was 0.05. In Feline Patient 2 (Himalayan), which underwent extirpation of ductal carcinoma on October 17, 2006, the absorbance at 450 nm was 0.34. On the other hand, the

0 absorbance was not detected in healthy cats at all.

[0318]

Thus, similarly to dogs, the absorbance value was detected in samples from cats suffering from cancer, while the absorbance value was not detected at all in samples from healthy cats. Hence, similarly to dogs, cancers in cats can also be

5 detected by this method.

[0319] (4) Diagnosis in Healthy Human

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2017239516 04 Oct 2017

Using the above-described partial polypeptide of canine TRIP 11 and antihuman IgG antibody above, the IgG antibody titer of healthy human serum which specifically reacts with the polypeptide was measured in the same manner as described above. As a secondary antibody, HRP-conjugated anti-human IgG antibody (HRP-Goat Anti-Human IgG(H+L) Conjugate: manufactured by Zymed Laboratories) 10000-fold diluted with the blocking solution was used. As a positive control, an immobilized ovalbumin antigen prepared by immobilizing 50 pg/ml ovalbumin in phosphate buffered saline on a solid phase was used. As a result, in Healthy Human 1, the absorbance at 450 nm observed on an ovalbumin antigen was

0.25, while the absorbance at 450 nm observed on the recombinant protein was 0, not detected at all. Similarly, in Healthy Human 2, the absorbance at 450 nm observed on an ovalbumin antigen was 0.18, while the absorbance at 450 nm observed on the recombinant protein was 0, not detected at all.

[0320]

Further, the diagnosis was carried out in the same manner as described above using a full-length canine TRIP11 having the sequence shown in SEQ ID NO:45 prepared in Example D-2. Asa result, it was revealed that the diagnosis can be similarly attained in humans, dogs and cats.

[0321]

0 Example D-4: Cancer Diagnosis Using Polypeptide Derived from Human TRIP11

Using the partial polypeptide of human TRIP11 (SEQ ID NO:55; 236th to 1023rd amino acid region of SEQ ID NO:47) prepared in Example D-2, the IgG antibody titer of human, canine and feline sera which react with the polypeptide was measured in the same manner as in Example D-3.

[0322]

The diagnosis was carried out using healthy human serum. In the same manner as in Example D-3 (4), ovalbumin antigen was used as a positive control.

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As a result, the absorbance value was detected in the case where ovalbumin was immobilized on a solid phase, while the absorbance value was hardly detected in the case where the partial polypeptide of human TRIP11 was immobilized on a solid phase.

[0323]

Similarly, in healthy dogs and cats, the absorbance at 450 nm was hardly detected in the case where the polypeptide was immobilized on a solid phase. [0324]

On the other hand, Canine Patient 11 (Shih Tzu) underwent extirpation of mammary adenocarcinoma on June 21, 2007. According to the pathological diagnosis using the extirpated tissue, the mammary gland tissue contained highly atypical, invasive cells, and grew to form adenomatous hyperplasia showing large and small massive structures. Hence, this patient was diagnosed as malignant tumor. In Canine Patient 11, the absorbance at 450 nm was 0.19. The malignancy diagnosis was carried out using additional 310 serum samples which had been diagnosed as malignant by pathological diagnosis. As a result, by diagnosing a sample showing twice the average value of healthy canine samples as malignant, 74 samples i.e. 23.8% of the malignant cases could be successfully diagnosed as malignant. Moreover, in Feline Patient 3 (Mixed Breed), which underwent

0 extirpation of mammary adenocarcinoma on April 3,2007, the absorbance at 450 nm was 0.06.

[0325]

The above described results indicated that the diagnosis can also be similarly attained in humans, dogs and cats by using a polypeptide derived from human

TRIPll.

[0326]

Furthermore, pleural effusion and ascites samples collected from terminal

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2017239516 04 Oct 2017 cancer dogs were subjected to the diagnosis using the recombinant human protein in the same manner as the recombinant canine protein. As a result, values similar to those detected in serum samples could be detected, and hence cancer diagnosis could be successfully attained.

[0327]

In addition, the diagnosis was carried out in the same manner as described above using a full-length human TRIP11 having the sequence shown in SEQ ID NO:47 prepared in Example D-2. As a result, it was revealed that the diagnosis can also be similarly attained in humans, dogs and cats.

[0328]

Example D-5: Cancer Diagnosis by Measuring Antigen Polypeptide (1)

Mice and rabbits were immunized with the recombinant canine protein having the sequence shown in SEQ ID NO :54 prepared in Example D-2 to obtain an antibody specific to this antigen. Using this polyclonal antibody, detection of the 15 antigen polypeptide per se contained in the serum from cancer bearing living body was carried out by sandwich ELISA. Using anti-mouse IgG antibody, the amount of the protein in the serum which specifically reacts with the prepared polyclonal antibody specific to the prepared protein was measured by sandwich ELISA.

[0329]

As for immobilization of a primary antibody on a solid phase, 100 μ L/well of the rabbit antiserum 20-fold diluted with phosphate buffered saline was added to a 96-well Immobilizer Amino plate (manufactured by Nunc), and the plate was shaken at room temperature for 2 hours. As for blocking, 100 pL/well of 50 mM sodium bicarbonate buffer (pH 8.3) containing 0.5% BSA (bovine serum albumin,

5 manufactured by Sigma Aldrich Japan) (hereinafter referred to as blocking solution) was added to the plate, and the plate was shaken at room temperature for 1 hour. To the plate, 100 pL/well of the serum from cancer-bearing body diluted with the

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2017239516 04 Oct 2017 blocking solution was added, and the plate was shaken at room temperature for 3 hours to allow the reaction to proceed. As for the diluted serum, a 10-fold serial dilution ranging 10 to 1,000-fold was prepared. After washing the wells 3 times with phosphate buffered saline containing 0.05% Tween20 (manufactured by Wako

Pure Chemicals) (hereinafter referred to as PBS-T), 100 pL/well of mouse antiserum 200-fold diluted with the blocking solution was added thereto, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 pL/wcll of HRP-conjugated mouse IgG antibody (Stabilized Goat Anti Mouse HRP conjugated : manufactured by PIERCE)

2000-fold diluted with the blocking solution was added thereto as a tertiary antibody, and the plate was shaken at room temperature for 1 hour to allow the reaction to proceed. After washing the wells 3 times with PBS-T, 100 μΐ/well of a HRP substrate, TMB (1-Step Turbo TMB (tetramethylbenzidine), manufactured by PIERCE), was added thereto, and the enzyme-substrate reaction was allowed to proceed at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding 100 μΐ/wel! of 0.5 M sulfuric acid solution (manufactured by Sigma Aldrich Japan), and then the absorbance was measured at 450 nm with a microplate reader. As a control, a plate on which the rabbit antiserum was not immobilized and a plate with which serum from a cancer-bearing body was not reacted were

0 measured in the same manner as described above.

[0330]

As a result, the polypeptide was detected in cancer-bearing dogs and cats suffering from cutaneous leiomyosarcoma, breast cancer, malignant melanoma or the like, while the polypeptide was not detected in healthy dogs, healthy cats and healthy 2 5 humans. Hence, cancers could also be diagnosed by this method in which the antigen polypeptide was detected with an antibody which was prepared by using the recombinant canine polypeptide as an immunogen.

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2017239516 04 Oct 2017 [0331]

In addition, the diagnosis was carried out in the same manner as described above using an antibody which was prepared by using as an immunogen the fulllength canine TRIP11 having the sequence shown in SEQ ID NO:45 prepared in

Example D-2.

[0332]

As a result, cancers could also be diagnosed in dogs and cats by this method in which the antigen polypeptide was detected with an antibody which was prepared by using a full-length canine TRIP11 as an immunogen.

[0333]

Example D-6: Cancer Diagnosis by Measuring Antigen Polypeptide (2)

Mice and rabbits were immunized with the recombinant human protein having the sequence shown in SEQ ID NO:55 prepared in Example D-2 to obtain an antibody specific to this antigen. In the same manner as in Example D-5, detection 15 of the antigen polypeptide per se contained in the serum from cancer-bearing body was carried out by sandwich ELISA using this polyclonal antibody.

[0334]

As a result, the polypeptide was detected in cancer-bearing dogs and cats suffering from cutaneous leiomyosarcoma, breast cancer, malignant melanoma or the 2 0 like, while the polypeptide was not detected in healthy dogs, healthy cats and healthy humans. Hence, cancers could also be diagnosed by this method in which the antigen polypeptide was detected with an antibody prepared by using the recombinant human polypeptide as an immunogen.

[0335]

In addition, the diagnosis was carried out in the same manner as described above using an antibody which was prepared by using as an immunogen the fulllength human TRIP11 having the sequence shown in SEQ ID NO:47 prepared in

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2017239516 04 Oct 2017

Example D-2.

[0336]

As a result, cancers could also be diagnosed in dogs and cats by this method in which the antigen polypeptide was detected with an antibody which was prepared 5 by using a full-length human TRIP11 as an immunogen.

[0337]

Example E-l: Combined Diagnosis of Cancer Using Four Antigen Polypeptides (1) (1) Cancer Diagnosis in Dogs

Using the recombinant canine polypeptide (SEQ ID NO:2) prepared in

Example A-2, the canine calmegin protein (SEQ ID NO: 16) prepared in Example B2, the full-length (SEQ ID NO:26 or 42) or partial (SEQ ID NO:35; 1514th to 2339th amino acid region of SEQ ID NO :26) polypeptide of canine CEP prepared in Example C-2, the full-length (SEQ ID NO:45) or partial (SEQ ID NO:54; 237th to 1023rd amino acid region of SEQ ID NO:45) polypeptide of canine TRIP11 prepared in Example D-2 and anti-dog IgG antibody, the IgG antibody titer of the serum which specifically reacts with any of the proteins or polypeptides mentioned above was measured.

[0338]

By diagnosing a sample showing twice the average value of healthy canine

0 samples as malignant, 272 samples i.e. 87.5% of the malignant cases could be successfully diagnosed as malignant. The subject living body was diagnosed as malignant when any one of 4 proteins and polypeptides indicated malignancy (the same shall apply hereinafter). The details of these 272 cancer samples are as follows. It is noted that the following number of each cancer case is a cumulative

5 total, as some samples contained multiple primaries.

Malignant melanoma, 10 cases; lymphoma, 13 cases; pheochromocytoma, 1 case; suppurative inflammation, 1 case; granulosa cell tumor, 1 case; hepatocellular

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2017239516 04 Oct 2017 carcinoma, 5 cases; angioma, 1 case; malignant testicular tumor, 8 cases; intraoral tumor, 4 cases; perianal adenocarcinoma, 14 cases; osteosarcoma, 5 cases; fibrosarcoma, 9 cases; ductal carcinoma, 10 cases; chondrosarcoma, 2 cases; mammary adenocarcinoma, 56 cases; combined mammary adenocarcinoma, 26 cases; lung cancer, 2 cases; sebaceous carcinoma, 2 cases; nasal adenocarcinoma, 2 cases; mastocytoma, 37 cases; adrenomedullary tumor, 1 case; leiomyosarcoma, 2 cases; squamous cell carcinoma, 11 cases; chronic lymphocytic leukemia, 1 case; undifferentiated sarcoma, 2 cases; malignant mixed tumor, 2 cases; tumor in the posterior segment of the left lobe of the lung, 1 case; tumor in the right infra-axillary region, 1 case; tumor in the elbow of the right forelimb, 1 case; bladder cancer (transitional cell carcinoma), 1 case; metastatic malignant melanoma, 3 cases; amelanotic malignant melanoma, 1 case; adenocarcinoma of the large intestine, 1 case; plasmacytoma, 1 case; histiocytic sarcoma, 1 case; liposarcoma, 1 case; poorly differentiated sarcoma, 1 case; synovial sarcoma, 1 case; malignant hemangiopericytoma, 1 case; apocrine sweat gland carcinoma, 3 cases; bronchial adenocarcinoma, 1 case; germinoma, 1 case; malignant fibrous histiocytoma, 1 case; metastatic malignant epithelioma, 1 case; mammary ductal carcinoma, 1 case; angiosarcoma, 1 case; tubular mammary adenocarcinoma, 1 case; invasive trichoepithelioma, 1 case; prostate cancer, 1 case; soft part sarcoma (spindle cell

0 tumor), 1 case; ceruminous adenocarcinoma, 1 case; multicentric lymphoma, 2 cases; invasive trichoepithelioma, 1 case; anal sac adenocarcinoma, 1 case; apocrine carcinoma, 1 case; gastric adenocarcinoma, 1 case; seminoma, 1 case; basal cell carcinoma, 1 case; hemangiopericytoma, 4 cases; myxosarcoma, 1 case; sebaceous epithelioma, 1 case; splenic tumor, 1 case.

[0339] (2) Cancer Diagnosis in Cats

Next, cancer-bearing cats and healthy cats were diagnosed. Using 4 kinds of

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2017239516 04 Oct 2017 canine antigen polypeptides described above and anti-cat IgG antibody, the IgG antibody titer of feline serum which specifically reacts with any of the polypeptides was measured in the same manner as described above. As a secondary antibody, HRP-conjugated anti-cat IgG antibody (PEROXIDASE-CONJUGATED GOAT IgG 5 FRACTION TO CAT IgG (WHOLE MOLECULE): manufactured by CAPPEL

RESERCH REAGENTS) 8,000-fold diluted with the blocking solution was used. [0340]

Among a total of 17 samples used in the cancer diagnosis, 11 samples were definitely diagnosed as malignant by the pathological diagnosis using the extirpated tumor tissue. By diagnosing a sample showing twice the average value of healthy feline samples as malignant, 9 samples i.e. 81.8% of the malignant cases could be successfully diagnosed as malignant [0341]

Example E-2: Combined Diagnosis of Cancer Using Four Antigen Polypeptides (2) (1) Cancer Diagnosis in Dogs

Using the recombinant human polypeptide (SEQ ID NO:4) prepared in

Example A-2, the human calmegin protein (SEQ ID NO: 18) prepared in Example B2, the full-length (SEQ ID NO:28) or partial (SEQ ID NO:36; 1513rd to 2325th amino acid region of SEQ ID NO:28) polypeptide of human CEP prepared in

0 Example C-2, the full-length (SEQ ID NO:47) or partial (SEQ ID NO:55; 236th to

1023rd amino acid region of SEQ ID NO :47) polypeptide of human TRIP 11 prepared in Example D-2 and anti-dog IgG antibody, the IgG antibody titer of the serum which specifically reacts with any of the proteins or polypeptides mentioned above was measured in the same manner as described above.

[0342]

By diagnosing a sample showing twice the average value of healthy canine samples as malignant, 268 samples i.e. 86.2% of the malignant cases could be

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2017239516 04 Oct 2017 successfully diagnosed as malignant.

[0343] (2) Cancer Diagnosis in Cats

Next, cancer-bearing cats and healthy cats were diagnosed. Using 4 kinds of canine antigen polypeptides described above and anti-cat IgG antibody, the IgG antibody titer of feline serum which specifically reacts with any of the polypeptides was measured in the same manner as described above. As a secondary antibody, HRP-conjugated anti-cat IgG antibody (PEROXIDASE-CONJUGATED GOAT IgG FRACTION TO CAT IgG (WHOLE MOLECULE): manufactured by CAPPEL

RESERCH REAGENTS) 8,000-fold diluted with the blocking solution was used. [0344]

Among a total of 17 samples used in the cancer diagnosis, 11 samples were definitely diagnosed as malignant by the pathological diagnosis using the extirpated tumor tissue. By diagnosing a sample showing twice the average value of healthy feline samples as malignant, 7 samples i.e. 63.6% of the malignant cases could be successfully diagnosed as malignant.

[0345]

Example E-3: Combined Diagnosis of Cancer by Measuring Four Antigen Polypeptides (1)

0 Mice and rabbits were immunized with the recombinant canine polypeptide (SEQ ID NO:2) prepared in Example A-2, the canine calmegin protein (SEQ ID NO: 16) prepared in Example B-2, the full-length (SEQ ID NO:26 or 42) or partial (SEQ ID NO:35; 1514th to 2339th amino acid region of SEQ ID NO:26) polypeptide of canine CEP prepared in Example C-2, or the full-length (SEQ ID NO:45) or

5 partial (SEQ ID NO:54; 237th to 1023rd amino acid region of SEQ ID NO:45) polypeptide of canine TRIP11 prepared in Example D-2 to obtain specific antibodies against these antigens. In the same manner as in Examples A, B, C, D-5, the

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2017239516 04 Oct 2017 antigen polypeptides per se contained in serum from cancer-bearing living body were detected by sandwich ELISA using the prepared polyclonal antibodies.

[0346]

As a result, this method in which antigen polypeptides were detected using antibodies prepared by using canine antigen polypeptides as an immunogen could successfully diagnose 252 samples i.e. 81.0% of the malignant cases as malignant by diagnosing a sample showing twice the average value of healthy canine samples as malignant. Similarly, also in cats, 8 samples i.e. 72.7% of the malignant cases could be successfully diagnosed as malignant by diagnosing a sample showing twice the average value of healthy feline samples as malignant.

[0347]

Example E-4: Combined Diagnosis of Cancer by Measuring Four Antigen Polypeptides (2)

Mice and rabbits were immunized with the recombinant human polypeptide (SEQ ID NO:4) prepared in Example A-2, the human calmegin protein (SEQ ID

NO:18) prepared in Example B-2, the full-length (SEQ ID NO:28) or partial (SEQ ID NO:36; 1513rd to 2325th amino acid region of SEQ ID NO:28) polypeptide of human CEP prepared in Example C-2, or the full-length (SEQ ID NO:47) or partial (SEQ ID NO:55; 236th to 1023rd amino acid region of SEQ ID NO:47) polypeptide of human TRIP 11 prepared in Example D-2 to obtain specific antibodies against these antigens. In the same manner in Examples A, B, C, D-5, the antigen polypeptides per se contained in serum from cancer-bearing living body were detected by sandwich ELISA using the prepared polyclonal antibodies.

[0348]

As a result, this method in which antigen polypeptides were detected using antibodies prepared by using human antigen polypeptides as an immunogen could successfully diagnose 248 samples i.e. 79.7% of the malignant cases as malignant by

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2017239516 04 Oct 2017 diagnosing a sample showing twice the average value of healthy canine samples as malignant. Similarly, also in cats, 7 samples i.e. 63.6% of the malignant cases could be successfully diagnosed as malignant by diagnosing a sample showing twice the average value of healthy feline samples as malignant.

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Claims (18)

1. A method for detecting a cancer(s), which is applied to a sample separated from a living body and comprises measuring an expression of a polypeptide having a reactivity to bind to an antibody against a centrosomal protein having the amino acid sequence shown in SEQ ID NO:26, 28 or 42 by antigen-antibody reaction.

2. The method according to claim 1, wherein the polypeptide is a centrosomal protein having the amino acid sequence shown in SEQ ID NO:26 or 42 or a polypeptide having a homology of not less than 95% thereto.

3. The method according to claim 1 or 2, wherein said living body is a dog, a human or a cat.

4. The method according to claim 3, wherein said living body is a dog and wherein said polypeptide to be measured is a polypeptide having the amino acid sequence shown in SEQ ID No:26.

5. The method according to claim 3, wherein said living body is a human and wherein said polypeptide to be measured is a polypeptide having the amino acid sequence shown in SEQ ID No:28.

6. The method according to any one of claims 1 to 5, wherein measurement of the expression of said polypeptide is carried out by measuring an antibody which may be contained in the sample by immunoassay, said antibody being induced in the living body against said polypeptide to be measured.

7. The method according to claim 6, which is carried out by immunoassay using as an antigen a polypeptide consisting of not less than 500 consecutive amino acids of the amino acid sequence

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2017239516 10 Apr 2019 shown in SEQ ID NO:26 and comprising not less than 500 consecutive amino acids located in the region of 1514th to 2339th amino acids of SEQ ID NO:26, or a polypeptide consisting of not less than 500 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:28 and comprising not less than 500 consecutive amino acids located in the region of 1513rd to 2325th amino acids of SEQ ID NO:28.

8. The method according to claim 7, wherein the polypeptide comprises a region of 1514th to 2339th amino acids of the amino acid sequence shown in SEQ ID NO:26 and consists of not more than 1000 amino acids, or comprises a region of 1513rd to 2325th amino acids of the amino acid sequence shown in SEQ ID NO :28 and consists of not more than 1000 amino acids

9. The method according to claim 8, wherein the polypeptide has the amino acid sequence shown in SEQ ID NO:35 or 36.

10. The method according to any one of claims 1 to 5, wherein measurement of the expression the polypeptides is carried out by measuring said polypeptide which may be contained in the sample by immunoassay.

11. The method according to any one of claims 1 to 10, wherein said sample is a serum, plasma, ascites or a pleural effusion.

12. The method according to any one of claims 1 to 5, wherein measurement of the expression of the polypeptides is carried out by measuring mRNA encoding said polypeptide, which mRNA may be contained in the sample.

13. The method according to any one of claims 1 to 12, further comprising detecting a grade of cancer malignancy based on the expression level of the polypeptide, wherein a higher expression level of said polypeptide indicates a higher grade of malignancy.

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14. The method according to any one of claims 1 to 13, further comprising detecting stage of cancer progression based on the expression level of the polypeptide, wherein a higher expression level of said polypeptide indicates a more advanced stage.

15. The method according to any one of claims 1 to 14, further comprising monitoring an effect of treatment of said cancer(s) based on whether the expression level of the polypeptide decreases or not.

16. A reagent when used for detecting a cancer(s), comprising a polypeptide which immunologically reacts with an antibody induced in a living body against a polypeptide having the amino acid sequence shown in SEQ ID NO:26, 28 or 42.

17. A reagent when used for detecting a cancer(s), comprising an antibody which immunologically reacts with a polypeptide having a reactivity to bind to an antibody against a centrosomal protein having the amino acid sequence shown in SEQ ID NO:26, 28 or 42 by antigenantibody reaction, or antigen-binding fragment thereof.

18. A reagent when used for detecting a cancer(s), comprising a polynucleotide which specifically hybridizes with a partial region of the base sequence shown in any one of SEQ ID NOs:25, 27 and41.

2017239516 04 Oct 2017