Cancer diagnosis application of p53 isomer mutant

Abstract

A method for collecting data for cancer diagnosis, onset risk determination, malignancy determination, and / or prognosis prediction according to the present invention comprises (a) a step for causing an antigen-antibody reaction by bringing a sample from a subject into contact with a tumor-associated antigen; (b) a step for collecting data for cancer diagnosis, onset risk determination, malignancy determination, and / or prognosis prediction, (c) a step for comparing the measured amount of anti-p53 antibody with a predetermined reference amount of anti-p53 antibody for a tumor-associated antigen, and (c) a step for measuring the amount of anti-p53 antibody in a sample that specifically binds to the tumor-associated antigen; the tumor-associated antigen includes one or more p53 isomer mutants comprising an amino acid sequence having a mutation in any one of amino acid sequences of SEQ ID NO: 1-3, and the mutation includes any one of an N-terminal deletion mutation, a C-terminal deletion mutation, and a deletion mutation of both the N-terminal and the C-terminal.

Description

Technical Field

[0001] The present invention relates to a method for collecting data for diagnosis, onset risk assessment, malignancy assessment and / or prognosis prediction of cancer using p53 isoform mutants, and a kit for cancer diagnosis. Background Art

[0002] TP53 is the most famous gene in cancer research. Specifically, it is known that normal p53 protein is responsible for important tasks such as DNA repair, apoptosis induction, and cell senescence to inhibit cancer. However, it is generally believed that once certain mutations occur in TP53, the resulting mutants of p53 protein may lead to the formation of malignant tumors. For example, it is believed that mutants of p53 protein may promote cell proliferation or may also cause cancer metastasis. In addition, it is known that there are multiple isomers of the translation product of TP53. Specifically, as isomers of TP53, in addition to the wild-type p53 isomer A, there are also p53 isomers B, C, G, I, H, D, E, F, J, K and L (refer to non-patent literature 1 and non-patent literature 2) with C-terminal structural differences caused by alternative splicing or N-terminal deletion mutations caused by abnormal splicing. However, there are many unknowns in the functions of each isomer.

[0003] On the other hand, in recent years, there have been many reports on methods of using autoantibodies as markers of diseases or disease sensitivity. For example, Patent Document 1 describes a method in which when the level of autoantibodies that specifically bind to tumor marker proteins such as MUC1, MUC16, c-myc, EGFR, and p53 in a sample collected from a subject increases, it can be determined that the subject may have a risk of developing cancer.

[0004] In addition, as a cancer diagnosis auxiliary reagent for measuring the expression level of autoantibodies (anti-p53 antibodies) against p53 protein in human serum or plasma, the "MESACUP (registered trademark) anti-p53test" manufactured by MBL (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.) is known. Specifically, this reagent uses wild-type p53 isoform A to measure the expression level of anti-p53 antibodies in serum or plasma. Anti-p53 antibodies appear with high specificity in malignant tumors, and when used in combination with other cancer markers, there is little overlap in the positive rates for different types of cancer. Therefore, by combining anti-p53 antibodies with a variety of other cancer markers and measuring their values, the detection rate of various cancers such as esophageal cancer, colorectal cancer, and breast cancer can be efficiently improved.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2014-16364

[0008] Non-patent literature

[0009] Non-patent document 1: Jean-Christophe Bourdon et al., “p53 isoforms can regulate p53 transcriptional activity”, GENES & DEVELOPMENT, 2005, 19: 2122-2137

[0010] Non-patent document 2: V Marcel et. al., "Biological functions of p53 isoforms through evolution: lessons from animal and cellular models", Cell Death and Differentiation (2011) 18, 1815-1824 Summary of the invention

[0011] An object of the present invention is to provide a method for collecting data for diagnosis, risk assessment, malignancy assessment and / or prognosis prediction of cancer with good sensitivity while maintaining high specificity.

[0012] The present inventors have conducted intensive studies to solve the above-mentioned problems and have completed the present invention.

[0013] The method of the first aspect of the present invention is a method for collecting data for cancer diagnosis, risk assessment, malignancy assessment and / or prognosis prediction, comprising:

[0014] (a) a step of inducing an antigen-antibody reaction by contacting a sample from a subject with one or more tumor-associated antigens;

[0015] (b) a step of measuring the amount of the anti-p53 antibody in the sample that specifically binds to the one or more tumor-associated antigens; and

[0016] (c) comparing the measured amount of the anti-p53 antibody with a predetermined reference amount of the anti-p53 antibody for the one or more tumor-associated antigens;

[0017] The above-mentioned tumor-associated antigen includes one or more p53 isoform mutants consisting of an amino acid sequence having a mutation in any one of the amino acid sequences of SEQ ID NOs: 1 to 3, and the above-mentioned mutation includes any one of an N-terminal deletion mutation, a C-terminal deletion mutation, and a deletion mutation of both the N-terminus and the C-terminus.

[0018] The cancer diagnostic kit of the second aspect of the present invention comprises one or more p53 isoform mutants consisting of an amino acid sequence having a mutation in any one of the amino acid sequences of SEQ ID NOs: 1 to 3, and the above mutation comprises any one of an N-terminal deletion mutation, a C-terminal deletion mutation, and a deletion mutation of both the N-terminus and the C-terminus. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram showing p53 isoforms A to L.

[0020] Figure 2 Schematic diagram showing the locations of nonsense mutations in various p53 isoform mutants. DETAILED DESCRIPTION

[0021] As described above, regarding the use of anti-p53 antibodies in the sample as cancer markers, anti-p53 antibodies are useful because they have high specificity for malignant tumors. However, although anti-p53 antibodies have high specificity for a variety of cancers, there is a problem of low sensitivity in detecting cancer. In other words, for example, when anti-p53 antibodies are used as cancer markers, when they are judged to be positive, the possibility that the subject has cancer is high. However, even when they are judged to be negative, it cannot be said that the possibility that the subject has cancer is necessarily low. Therefore, not only because of the small overlap in the positive rate of other cancer markers for cancer types, but also because of the low sensitivity of detecting cancer, anti-p53 antibodies are usually used in combination with other cancer markers when used as cancer markers.

[0022] Therefore, when using an anti-p53 antibody as a cancer marker, it would be very useful if cancer could be detected with high sensitivity while maintaining the high specificity peculiar to the anti-p53 antibody.

[0023] At present, when anti-p53 antibodies are used as cancer markers, wild-type p53 isoform A consisting of the amino acid sequence of sequence number 1 is often used as an antigen for inducing an antigen-antibody reaction. Furthermore, as the type of antibody isotype, IgG antibodies are generally used. The present inventors have repeatedly conducted various studies on methods that can detect cancer with better sensitivity while maintaining the high specificity peculiar to anti-p53 antibodies compared to the conventional general methods. As a result, the following insights described in (1) to (3) were discovered, thereby completing the present invention.

[0024] (1) In human plasma, there are a variety of anti-p53 IgG antibodies that specifically bind to each p53 isoform mutant and cannot be recognized by wild-type p53 isoform A. Furthermore, such specific anti-p53 antibodies are present in significantly higher amounts in p53 isoform mutants having N-terminal deletion mutations, C-terminal deletion mutations, or deletion mutations at both the N-terminus and the C-terminus. In other words, it is speculated that by performing an assay using anti-p53 antibodies for each p53 isoform mutant having such mutations and combining the results, the sensitivity of cancer detection can be improved.

[0025] (2) In human plasma, there are anti-p53 IgG antibodies that specifically bind to p53 isoform C and cannot be recognized by wild-type p53 isoform A. In addition, there may also be anti-p53 IgG antibodies that specifically bind to p53 isoform B. Therefore, it is speculated that by performing an assay using anti-p53 antibodies against p53 isoform B, p53 isoform C, and / or the above-mentioned p53 isoform mutants and combining these assay results, the sensitivity of cancer detection can be improved.

[0026] (3) Regarding the measurement results of anti-p53 antibodies that specifically bind to each p53 isoform mutant, the positive detection results obtained using IgG antibodies are different from the positive detection results obtained using IgA antibodies. Furthermore, regarding the measurement results of anti-p53 antibodies that specifically bind to p53 isoforms A to C, the positive detection results obtained using IgG antibodies are different from the positive detection results obtained using IgA antibodies. Therefore, it is speculated that by using p53 isoforms A to C and p53 isoform mutants to measure isotype antibodies other than the IgG antibodies that are currently commonly measured, the sensitivity of cancer detection can be improved. It is further speculated that by combining the positive detection results of anti-p53 antibodies obtained using two or more antibodies (preferably two or more antibodies including IgG antibodies and IgA antibodies), the sensitivity of cancer detection can be improved.

[0027] Based on the insights of (1) to (3) above, the following conclusions can be obtained. By using one or more of p53 isoform B, p53 isoform C, and p53 isoform mutants having N-terminal deletion mutations (p53 isoforms G, I, H, D, E, F, J, K, and L), C-terminal deletion mutations, and deletion mutations of both N-terminal and C-terminal ends as antigens for inducing antigen-antibody reactions, anti-p53 antibodies that cannot be identified by conventional methods can be widely identified. As a result, it is speculated that cancer can be detected with better sensitivity. It is also speculated that by also using wild-type p53 isoform A as an antigen, cancer can be detected with better sensitivity.

[0028] It is also speculated that by using one or more of p53 isoforms A to C and p53 isoform mutants having the above-mentioned deletion mutations as antigens and detecting one or more antibodies of isotypes other than IgG antibodies or two or more antibodies of all isotypes including IgG antibodies, cancer can be detected with better sensitivity.

[0029] Therefore, using the findings from (1) to (3) above, a method can be obtained for collecting various data for cancer diagnosis, onset risk assessment, malignancy assessment and / or prognosis prediction with good sensitivity while maintaining the high specificity of anti-p53 antibodies as cancer markers.

[0030] That is, according to the present invention, a method for collecting data for diagnosis, onset risk determination, malignancy determination and / or prognosis prediction of cancer with good sensitivity while maintaining high specificity can be provided.

[0031] <Definition>

[0032] In the present specification, "wild-type p53 isoform A" mainly refers to a protein consisting of the amino acid sequence of SEQ ID NO: 1. However, "wild-type p53 isoform A" may contain some mutations such as addition, deletion and / or substitution of amino acids as described below, as long as its specific binding to anti-p53 antibodies is at the same level as that of the protein of SEQ ID NO: 1.

[0033] In this specification, "p53 isomer B" mainly refers to a protein consisting of the amino acid sequence of sequence number 2. However, in "p53 isomer B", as long as its specific binding to p53 antibodies is at the same level as that of the protein of sequence number 2, it may include a part of mutations such as addition, deletion and / or substitution of amino acids as described later. In addition, in this specification, "p53 isomer C" mainly refers to a protein consisting of the amino acid sequence of sequence number 3. However, in "p53 isomer C", as long as its specific binding to p53 antibodies is at the same level as that of the protein of sequence number 3, it may include a part of mutations such as addition, deletion and / or substitution of amino acids as described later. It should be noted that p53 isomer B and p53 isomer C are isomers whose C-terminal structures differ due to selective splicing when compared with wild-type p53 isomer A.

[0034] In the present specification, "p53 isoform mutant having an N-terminal deletion mutation" refers to a p53 isoform mutant consisting of an amino acid sequence having an N-terminal deletion mutation caused by abnormal splicing in the amino acid sequence of SEQ ID NOs. 1 to 3. It should be noted that the p53 isoform mutant having an N-terminal deletion mutation may contain other mutations such as addition, deletion and / or substitution of amino acids as described below in addition to the deletion mutation, as long as it does not affect the specific binding of the anti-p53 antibody. Specific examples of such p53 isoform mutants having an N-terminal deletion mutation include p53 isoform G (sequence number 4), p53 isoform I (sequence number 5), p53 isoform H (sequence number 6), p53 isoform D (sequence number 7), p53 isoform E (sequence number 8), p53 isoform F (sequence number 9), p53 isoform J (sequence number 10), p53 isoform K (sequence number 11), and p53 isoform L (sequence number 12) (see Non-Patent Document 1 and Non-Patent Document 2).

[0035] In this specification, "p53 isoform mutant having a C-terminal deletion mutation" refers to a p53 isoform mutant consisting of an amino acid sequence having a C-terminal deletion mutation derived from a nonsense mutation in the amino acid sequence of sequence numbers 1 to 3. It should be noted that the p53 isoform mutant having a C-terminal deletion mutation may also contain other mutations such as addition, deletion and / or substitution of amino acids as described below other than the deletion mutation, as long as it does not affect its specific binding to anti-p53 antibodies. As such a p53 isoform mutant having a C-terminal deletion mutation, specifically, a protein consisting of any amino acid sequence in sequence numbers 13 to 18 can be cited.

[0036] In the present specification, "a p53 isoform mutant having a deletion mutation at both the N-terminus and the C-terminus" refers to a p53 isoform mutant consisting of an amino acid sequence having both an N-terminal deletion mutation caused by splicing abnormality and a C-terminal deletion mutation from a nonsense mutation in the amino acid sequence of sequence numbers 1 to 3. It should be noted that the p53 isoform mutant having a deletion mutation at both the N-terminus and the C-terminus may also contain other mutations such as addition, deletion and / or substitution of amino acids described below other than the deletion mutation as long as it does not affect its specific binding to anti-p53 antibodies. Specifically, "a p53 isoform mutant having a deletion mutation at both the N-terminus and the C-terminus" refers to a p53 isoform mutant consisting of an amino acid sequence having a C-terminal deletion mutation from a nonsense mutation in the amino acid sequence of sequence numbers 4 to 6, 7 to 9, or 10 to 12. Examples of such p53 isoform mutants having deletion mutations at both the N-terminus and the C-terminus include proteins consisting of any one of the amino acid sequences of SEQ ID NOs: 19 to 35.

[0037] In the present specification, the "anti-p53 antibody (or autoantibody against p53 protein)" refers to an antibody produced in the body of a subject, which is an object, and which targets the p53 protein present in the body of the subject.

[0038] In this specification, "diagnosing cancer, determining the risk of developing cancer, determining the malignancy and / or predicting the prognosis" includes the following meanings: determining or predicting whether the possibility of the subject having cancer is high; determining or predicting the risk of developing cancer in the subject; determining or predicting the malignancy, severity or progression of the subject's cancer; (for example, continuously) monitoring the treatment effect of the subject's cancer; and determining or predicting that the subject's cancer has a poor prognosis.

[0039] In the present specification, the term "cancer" is not particularly limited, and examples thereof include brain tumors, head and neck cancer, breast cancer, uterine cancer, cervical cancer, uterine sarcoma, ovarian cancer, esophageal cancer, salivary gland cancer, thyroid cancer, stomach cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor (GIST), mesothelioma, kidney cancer, small intestine cancer, lung cancer, small cell lung cancer, renal pelvis / ureter cancer, osteosarcoma, Ewing's sarcoma, chondrosarcoma, prostate cancer, testicular cancer, renal cell carcinoma, bladder cancer, rhabdomyosarcoma, skin cancer, anal cancer, mesothelioma, malignant lymphoma, leukemia, chronic lymphocytic leukemia (CLL), multiple myeloma, melanoma, and the like.

[0040] In this specification, "subject" mainly refers to humans and other mammals. Examples of other mammals include primates such as monkeys and chimpanzees; domestic animals such as cattle, horses, pigs, and sheep; pet animals such as dogs and cats; and experimental animals such as mice, rats, and rabbits. Among these, the subject is preferably a human.

[0041] In this specification, "sample (or sample from the subject)" is not particularly limited as long as it is a sample from a subject that is suitable for data collection for diagnosis of cancer, risk of onset determination, malignancy determination and / or prognosis prediction, and can induce antigen-antibody reaction by contact with an antigen. In addition, in this specification, "sample (or sample from the subject)" also includes a prepared sample prepared by diluents, etc., of body fluids directly collected from the subject. Furthermore, it is preferred to select a suitable sample according to the isotype of the antibody to be measured. As a sample, for example, whole blood, serum, plasma, urine, prostatic fluid, tears, mucous ascites, oral fluid, saliva, semen, seminal plasma, mucus, sweat, nasal mucus, vaginal fluid, stool, sputum, cerebrospinal fluid, bone marrow, lymph, fetal fluid (such as amniotic fluid), milk, etc. From the perspective of ease of collection, the sample preferably includes one or more of serum, plasma, oral fluid and saliva, and more preferably includes one or more of serum and plasma.

[0042] In this specification, "positive" means: the amount of anti-p53 antibodies as cancer markers in the sample is higher than a predetermined reference amount, so the possibility that the subject has cancer is high; the risk of cancer in the subject is high; the malignancy, severity or progression of the cancer in the subject is high; the therapeutic effect of the cancer in the subject is low; etc. In addition, in this specification, "negative" means: the amount of anti-p53 antibodies as cancer markers in the sample is lower than a predetermined reference amount, so the possibility that the subject has cancer is low; the risk of cancer in the subject is low; the malignancy, severity or progression of the cancer in the subject is low; the therapeutic effect of the cancer in the subject is high; etc.

[0043] The embodiments of the present invention are described in detail below. It should be noted that the scope of the present invention is not limited to the embodiments described here, and various modifications can be made within the scope that does not impair the gist of the present invention.

[0044] <Embodiment 1: Method for collecting data for diagnosis, onset risk assessment, malignancy assessment and / or prognosis prediction of cancer>

[0045] The method of embodiment 1 includes four methods. Specifically, it includes: a method related to the use of a p53 isoform mutant with a deletion mutation (embodiment 1-1); a method related to the use of any one of p53 isoform B, p53 isoform C, and a p53 isoform mutant with a deletion mutation (embodiment 1-2); a method related to the measurement of antibodies other than IgG antibodies (embodiment 1-3); and a method related to the measurement of two or more isotype antibodies (embodiment 1-4). The following is a detailed description.

[0046] (Implementation Method 1-1)

[0047] The method of embodiment 1-1 is a method of using an anti-p53 antibody that specifically binds to a p53 isoform mutant having a deletion mutation as a cancer marker. Specifically, the method of embodiment 1-1 includes (a) an antigen-antibody reaction step, (b) a step of measuring the amount of the anti-p53 antibody, and (c) a step of comparing the amount of the anti-p53 antibody. Each step is described in detail below.

[0048] (a) Antigen-antibody reaction step

[0049] In the antigen-antibody reaction step, an antigen-antibody reaction is induced by bringing a sample from a subject into contact with one or more tumor-associated antigens.

[0050] First, the tumor-associated antigen that comes into contact with the sample will be described in detail below.

[0051] The tumor-associated antigen includes any one or more of a p53 isoform mutant having an N-terminal deletion mutation, a p53 isoform mutant having a C-terminal deletion mutation, and a p53 isoform mutant having deletion mutations at both the N-terminus and the C-terminus (hereinafter, these mutants are also referred to as "p53 isoform mutants of Embodiment 1-1").

[0052] The p53 isoform mutant of Embodiment 1-1 is preferably a protein represented by any one of the following (i) to (iii).

[0053] (i) A protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 35.

[0054] (ii) A protein consisting of an amino acid sequence in which one or several amino acids are added, deleted and / or substituted in any one of the amino acid sequences of SEQ ID NOs: 4 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above has the same level of specific binding as the protein of the corresponding sequence number.

[0055] (iii) a protein consisting of an amino acid sequence having 95% or more homology to any one of the amino acid sequences of SEQ ID NOs: 4 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above and has the same level of specific binding as the protein of the corresponding sequence number.

[0056] In the p53 isoform mutant of embodiment 1-1, the protein consisting of the amino acid sequence of SEQ ID NOs. 4 to 35 in (i) above is a p53 isoform mutant having only one of an N-terminal deletion mutation, a C-terminal deletion mutation, and a deletion mutation of both the N-terminus and the C-terminus in any of the amino acid sequences of SEQ ID NOs. 1 to 3. Table 1 below summarizes the sequence numbers of the amino acid sequences of the protein of (i) above including the wild-type p53 isoform A, p53 isoform B, and p53 isoform C, the sequence numbers of the base sequences, specific mutations relative to the wild-type p53 isoform A, and the like.

[0057] [Table 1]

[0058]

[0059] in addition, Figure 1 The structures of wild-type p53 isoform A and p53 isoforms B, C, G, I, H, D, E, F, J, K, and L are schematically shown (see also Non-Patent Document 1 and Non-Patent Document 2). Figure 2 The positions of nonsense mutations of each p53 isoform mutant on the sequences of wild-type p53 isoform A and p53 isoforms B, C, G, I, H, D, E, F, J, K, and L are schematically shown.

[0060] like Figure 1 As shown, p53 isoform B (SEQ ID NO: 2) and p53 isoform C (SEQ ID NO: 3) differ in amino acid sequence from wild-type p53 isoform A in terms of C-terminal structure. p53 isoforms G (SEQ ID NO: 4), I (SEQ ID NO: 5), H (SEQ ID NO: 6), D (SEQ ID NO: 7), E (SEQ ID NO: 8), F (SEQ ID NO: 9), J (SEQ ID NO: 10), K (SEQ ID NO: 11), and L (SEQ ID NO: 12) each consist of an amino acid sequence having only an N-terminal deletion mutation in the amino acid sequence of p53 isoforms A to C.

[0061] Furthermore, as shown in Table 1 above, examples of p53 isoform mutants having only a C-terminal deletion mutation include p53 isoform mutants consisting of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. Figure 2As shown, the p53 isoform mutant consisting of the amino acid sequence of SEQ ID NO: 13 is a protein having a C-terminal deletion mutation in which the arginine at position 342 in the amino acid sequence of SEQ ID NO: 1 is replaced by a stop codon due to a nonsense mutation ( Figure 2 In the example, it is shown as "R342X". Figure 2 As shown, the p53 isoform mutant consisting of the amino acid sequence of SEQ ID NO: 14 is a protein having a C-terminal deletion mutation in which the glutamine at position 331 in any of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 3 is replaced by a stop codon due to a nonsense mutation ( Figure 2 , shown as "Q331X". ).

[0062] In addition, as shown in Table 1 above, examples of p53 isoform mutants having deletion mutations at both the N-terminus and the C-terminus include p53 isoform mutants consisting of the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. Figure 2 As shown, the p53 isoform mutant consisting of the amino acid sequence of SEQ ID NO: 19 is a protein having deletion mutations at both the N-terminus and the C-terminus, in which the arginine at position 303 in the amino acid sequence of SEQ ID NO: 4 having only an N-terminal deletion mutation is replaced by a stop codon due to a nonsense mutation ( Figure 2 In the example, it is shown as "R303X". Figure 2 As shown, the p53 isoform mutant consisting of the amino acid sequence of SEQ ID NO: 20 is a protein having deletion mutations at both the N-terminus and the C-terminus, in which the glutamine at position 292 in any of the amino acid sequences of SEQ ID NO: 4 to SEQ ID NO: 6 having only an N-terminal deletion mutation is replaced by a stop codon due to a nonsense mutation ( Figure 2 , shown as "Q292X". ).

[0063] As described in (ii) and (iii) above, the amino acid sequence of the p53 isoform mutant of Embodiment 1-1 may not be completely identical to any of SEQ ID NOs: 4 to 35.

[0064] In the present specification, "an anti-p53 antibody that specifically binds to a protein with a corresponding sequence number (or a specific sequence number) has the same level of specific binding as the protein with the corresponding sequence number" means, for example, that when the amount of anti-p53 antibodies in the same sample is measured using a protein microarray as described in detail in the examples described later, the normalized value is approximately the same as the value of the protein with the corresponding sequence number.

[0065] Furthermore, in the present specification, “one or several” is not particularly limited, and means, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0066] In this specification, "amino acid sequence having a homology of more than a specific value" is not particularly limited, and for example refers to an amino acid sequence having a homology of more than a specific value calculated using a method such as BLAST known to those skilled in the art. Furthermore, in this specification, "amino acid sequence having a homology of more than 95%" refers to an amino acid sequence having a homology of preferably more than 96%, more preferably more than 97%, further preferably more than 98%, and particularly preferably more than 99%.

[0067] In the method of embodiment 1-1, any one or more of the p53 isoform mutants of embodiment 1-1 described in (i) to (iii) (preferably (i)) can be used as a tumor-associated antigen for measuring anti-p53 antibodies as cancer markers. Thus, anti-p53 antibodies in samples that could not be identified by conventional methods can be identified. Therefore, data for cancer diagnosis, etc. can be collected with good sensitivity while maintaining high specificity.

[0068] Based on the results of the examples of the present inventors, the p53 isoform mutant of Embodiment 1-1 is more preferably a protein represented by any one of the following (I) to (III).

[0069] (I) A protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NOs: 16 to 24, SEQ ID NO: 26, and SEQ ID NOs: 29 to 35.

[0070] (II) A protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of the amino acid sequences of SEQ ID NOs: 4 to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16 to SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 29 to SEQ ID NO: 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) above has the same level of specific binding activity as the protein of the corresponding sequence number.

[0071] (III) a protein consisting of an amino acid sequence having 95% or more identity to any one of SEQ ID NOs: 4 to 6, 7, 8, 10, 11, 14, 16 to 24, 26, and 29 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) above has the same level of specific binding as the protein of the corresponding sequence number.

[0072] In the method of embodiment 1-1, any one or more of the p53 isoform mutants of embodiment 1-1 of (I) to (III) (preferably (I)) can be used as a tumor-associated antigen for measuring anti-p53 antibodies as cancer markers. As a result, anti-p53 antibodies in samples that could not be identified by conventional methods can be more reliably identified. Therefore, data for cancer diagnosis, etc. can be collected more reliably with good sensitivity while maintaining high specificity.

[0073] Furthermore, it is preferred to use two or more p53 isoform mutants of embodiment 1-1 as tumor-associated antigens in combination. Specifically, it is preferred to contact a sample from the same subject with two or more p53 isoform mutants of embodiment 1-1 to induce antigen-antibody reactions. At this time, including steps (b) to (c) described later, it is preferred to perform steps (a) to (c) on each of the p53 isoform mutants of embodiment 1-1 as tumor-associated antigens.

[0074] By using two or more p53 isoform mutants of embodiment 1-1 as tumor-associated antigens in combination, anti-p53 antibodies that specifically bind to each p53 isoform mutant can be widely identified. It should be noted that, for the p53 isoform mutants of embodiment 1-1 as tumor-associated antigens, it is more preferable to use three or more of them in combination, further preferably to use four or more of them in combination, and particularly preferably to use five or more of them in combination.

[0075] Furthermore, the tumor-associated antigen preferably also includes one or more p53 isoforms composed of any one of the amino acid sequences in sequence numbers 1 to 3. That is, as a tumor-associated antigen, it is preferred to use in combination one or more of wild-type p53 isoform A, p53 isoform B and p53 isoform C in addition to the p53 isoform mutant of embodiment 1-1. At this time, it is also preferred to contact the sample from the same subject with the p53 isoform mutant and each p53 isoform of embodiment 1-1 to induce antigen-antibody reactions respectively. Furthermore, as described above, it is preferred to perform steps (a) to (c) on the p53 isoform mutant and each p53 isoform of embodiment 1-1, including the steps (b) to (c) described later.

[0076] As a method for inducing an antigen-antibody reaction, any method known to those skilled in the art can be applied as long as the antigen-antibody reaction can be induced by contacting a sample from a subject containing anti-p53 antibodies with a tumor-associated antigen. Specifically, for example, any immunoassay known to those skilled in the art can be applied together with the subsequent step of measuring the amount of anti-p53 antibodies (b).

[0077] (b) Anti-p53 Antibody Amount Measurement Step

[0078] In the step of measuring the amount of the anti-p53 antibody, the amount of the anti-p53 antibody in the sample that specifically binds to one or more tumor-associated antigens is measured.

[0079] That is, in this step, the amount of the anti-p53 antibody as a cancer marker in the sample that specifically binds to the p53 isoform mutant of Embodiment 1-1 is measured.

[0080] When two or more p53 isoform mutants according to Embodiment 1-1 are used in combination as tumor-associated antigens, the amount of anti-p53 antibody in the sample that specifically binds to each p53 isoform mutant according to Embodiment 1-1 is measured. In addition, when not only the p53 isoform mutants according to Embodiment 1-1 but also one or more of wild-type p53 isoform A, p53 isoform B, and p53 isoform C are used in combination as tumor-associated antigens, the amount of anti-p53 antibody in the sample that specifically binds to the p53 isoform mutants according to Embodiment 1-1 and the amount of anti-p53 antibody in the sample that specifically binds to each p53 isoform are measured, respectively.

[0081] In this specification, "measurement" means preferably semi-quantitatively or quantitatively measuring the amount (or expression level) or concentration of anti-p53 antibodies in a sample from a subject. The amount may be an absolute amount or a relative amount to a negative control.

[0082] Furthermore, the isotype of the anti-p53 antibody is preferably one or more selected from the group consisting of IgG antibody, IgA antibody, IgM antibody and IgE antibody, more preferably two or more, and even more preferably 3 or more. In addition, the isotype of the anti-p53 antibody is more preferably one or more of IgG antibody and IgA antibody, and even more preferably a combination of IgG antibody and IgA antibody.

[0083] When there are two or more isotypes of the anti-p53 antibody, it is preferred to perform steps (b) to (c) for each type of anti-p53 antibody isotype, including the step (c) described later. It is speculated that by combining the results of the measured amounts of the anti-p53 antibody based on two or more isotypes, the sensitivity of cancer detection can be more efficiently improved after the subsequent step (c).

[0084] The amount of anti-p53 antibody as a cancer marker is not particularly limited, and any method known to those skilled in the art can be applied. For example, as a measurement method, any immunoassay known to those skilled in the art such as ELISA (Enzyme Linked Immunosorbent Assay), Western blotting, flow cytometry, multiplex assays based on Luminex (registered trademark), U-Plex, Focus array, etc., surface plasmon resonance, etc. can be applied.

[0085] (c) Anti-p53 antibody amount comparison step

[0086] In the step of comparing the amount of the anti-p53 antibody, the measured amount of the anti-p53 antibody is compared with a predetermined reference amount of the anti-p53 antibody for one or more tumor-associated antigens.

[0087] In the present specification, the "predetermined reference amount of anti-p53 antibodies to tumor-associated antigens" refers to the amount (or concentration, standardized numerical value, etc.) of anti-p53 antibodies that is predetermined based on the measurement method, the type of sample, the type of cancer, etc., and serves as an indicator of a high possibility that the subject has cancer, a high risk of developing cancer in the subject, a high degree of malignancy of the cancer in the subject, etc. Alternatively, the "predetermined reference amount of anti-p53 antibodies to tumor-associated antigens" may refer to the amount (or concentration, standardized numerical value, etc.) of anti-p53 antibodies that is predetermined based on the measurement method, the type of sample, the type of cancer, etc., and serves as an indicator of a low possibility that the subject has cancer, a low risk of developing cancer in the subject, a low degree of malignancy of the cancer in the subject, etc.

[0088] Such a reference amount can be determined, for example, based on the amount from a group of cancer patients measured using exactly the same method. The group of cancer patients can be, for example, a group diagnosed with cancer by a doctor, a group determined to have a high risk of developing cancer using a variety of other cancer markers, etc. Alternatively, such a reference amount can be determined, for example, based on the amount from a group of healthy people measured using exactly the same method. The group of healthy people can be, for example, a group diagnosed by a doctor as not having cancer, a group determined to have a low risk of developing cancer using a variety of other cancer markers, etc. The reference amount can vary according to various physiological parameters such as the type, age, and gender of the subject.

[0089] That is, in this step, the measured amount of anti-p53 antibody is compared with a reference amount that can be an indicator of cancer, thereby collecting data for diagnosis of cancer, determination of onset risk, determination of malignancy, and / or prediction of prognosis.

[0090] When two or more p53 isoform mutants of Embodiment 1-1 are used in combination as tumor-associated antigens, the measured amount of the anti-p53 antibody of each p53 isoform mutant of Embodiment 1-1 is compared with the reference amount. As a result, it is estimated that the sensitivity of cancer detection can be effectively improved by combining the determination results of the measured amount of the anti-p53 antibody widely recognized by using the p53 isoform mutants of each Embodiment 1-1.

[0091] Furthermore, when not only the p53 isoform mutant of Embodiment 1-1 but also one or more of wild-type p53 isoform A, p53 isoform B and p53 isoform C are used in combination as tumor-associated antigens, the measured amount of each anti-p53 antibody is compared with each reference amount. As a result, it is estimated that by combining the determination results of the measured amount of the anti-p53 antibody that is more widely recognized based on the use of the p53 isoform mutant of Embodiment 1-1 and each p53 isoform, the sensitivity of cancer detection can be improved more efficiently.

[0092] When two or more isotypes of anti-p53 antibodies are measured, the measured amounts of the anti-p53 antibodies of each isotype are compared with respective reference amounts. As a result, it is estimated that by combining the determination results of various isotypes, the sensitivity of cancer detection can be improved more efficiently.

[0093] In summary, according to the method of embodiment 1-1, data for cancer diagnosis, onset risk assessment, malignancy assessment and / or prognosis prediction can be collected with good sensitivity while maintaining the high specificity peculiar to anti-p53 antibodies.

[0094] (Implementation 1-2)

[0095] The method of embodiment 1-2 is a method of using an anti-p53 antibody that specifically binds to one or more of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1 as a cancer marker.

[0096] As described above, in conventional methods, when anti-p53 antibodies are used as cancer markers, wild-type p53 isoform A consisting of the amino acid sequence of SEQ ID NO: 1 is often used as an antigen.

[0097] However, according to the findings of the present inventors, it is speculated that there may be IgG antibodies that specifically bind to p53 isoform B (SEQ ID NO: 2) or p53 isoform C (SEQ ID NO: 3) and are anti-p53 antibodies that cannot be recognized by wild-type p53 isoform A. Therefore, according to the method of embodiment 1-2 described below, it is possible to identify anti-p53 antibodies in a sample that cannot be recognized by the conventional method. Therefore, data for cancer diagnosis, morbidity risk determination, malignancy determination and / or prognosis prediction can be collected with good sensitivity while maintaining high specificity.

[0098] The method of embodiment 1-2 is a method for collecting data for cancer diagnosis, risk assessment, malignancy assessment and / or prognosis prediction, which comprises:

[0099] (a) a step of inducing an antigen-antibody reaction by contacting a sample from a subject with one or more tumor-associated antigens;

[0100] (b) a step of measuring the amount of the anti-p53 antibody in the sample that specifically binds to the one or more tumor-associated antigens; and

[0101] (c) comparing the measured amount of the anti-p53 antibody with a predetermined reference amount of the anti-p53 antibody for the one or more tumor-associated antigens;

[0102] The tumor-associated antigen comprises one or more proteins selected from the group consisting of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1.

[0103] The p53 isoform B of Embodiment 1-2 may be a protein represented by any one of the following (i') to (iii').

[0104] (i') A protein consisting of the amino acid sequence of SEQ ID NO: 2.

[0105] (ii') A protein consisting of an amino acid sequence in which one or several amino acids are added, deleted and / or substituted in the amino acid sequence of SEQ ID NO: 2, and an anti-p53 antibody that specifically binds to the protein of SEQ ID NO: 2 has the same level of specific binding as the protein of SEQ ID NO: 2.

[0106] (iii') A protein consisting of an amino acid sequence having 95% or more homology to the amino acid sequence of SEQ ID NO: 2, and an anti-p53 antibody that specifically binds to the protein of SEQ ID NO: 2 has the same level of specific binding as the protein of SEQ ID NO: 2.

[0107] The p53 isoform C of Embodiment 1-2 may be a protein represented by any one of the following (i'') to (iii'').

[0108] (i'') A protein consisting of the amino acid sequence of SEQ ID NO: 3.

[0109] (ii'') a protein consisting of an amino acid sequence in which one or several amino acids are added, deleted and / or substituted in the amino acid sequence of SEQ ID NO: 3, and an anti-p53 antibody that specifically binds to the protein of SEQ ID NO: 3 has the same level of specific binding as the protein of SEQ ID NO: 3.

[0110] (iii'') a protein consisting of an amino acid sequence having 95% or more homology to the amino acid sequence of SEQ ID NO: 3, and an anti-p53 antibody that specifically binds to the protein of SEQ ID NO: 3 has the same level of specific binding as the protein of SEQ ID NO: 3.

[0111] That is, the method of embodiment 1-2 is the same as that of embodiment 1-1 above, except that at least one of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1 is used as a tumor-associated antigen for measuring an anti-p53 antibody as a cancer marker. Specifically, the definition of each term, the details of each step, the preferred embodiment, etc. are the same as those of embodiment 1-1 above.

[0112] For example, similarly to the above-mentioned embodiment 1-1, it is preferred to use two or more of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1 in combination as a tumor-associated antigen. Furthermore, similarly to the above-mentioned embodiment 1-1, as a tumor-associated antigen, not only one or more of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1 but also wild-type p53 isoform A consisting of the amino acid sequence of sequence number 1 may be used in combination. Furthermore, for example, similarly to the above-mentioned embodiment 1-1, the isotype of the anti-p53 antibody is preferably one or more selected from the group consisting of IgG antibody, IgA antibody, IgM antibody, and IgE antibody. When the isotype of the anti-p53 antibody is two or more, it is preferred to perform the above-mentioned steps (b) to (c) on each isotype of the anti-p53 antibody.

[0113] (Implementation 1-3)

[0114] The method of Embodiment 1-3 is a method for collecting data for diagnosis, onset risk determination, malignancy determination and / or prognosis prediction of cancer by measuring anti-p53 antibodies of isotypes other than IgG antibodies.

[0115] As described above, in conventional methods, IgG antibodies are generally used as the antibody isotype.

[0116] However, according to the knowledge found by the present inventors, it is speculated that the cancer detection results based on the amount of anti-p53 antibodies that specifically bind to any of the p53 isoforms A, p53 isoforms B, p53 isoforms C, and the p53 isoform mutants of embodiment 1-1 are different depending on the type of antibody isotype. Therefore, according to the method of embodiment 1-3 described below, it is also possible to widely identify anti-p53 antibodies in samples that cannot be identified by conventional methods. Therefore, data for cancer diagnosis, onset risk determination, malignancy determination, and / or prognosis prediction can be collected with good sensitivity while maintaining high specificity.

[0117] The method of embodiments 1-3 is a method for collecting data for cancer diagnosis, risk assessment, malignancy assessment and / or prognosis prediction, comprising:

[0118] (a) a step of inducing an antigen-antibody reaction by contacting a sample from a subject with one or more tumor-associated antigens;

[0119] (b) a step of measuring the amount of the anti-p53 antibody in the sample that specifically binds to the one or more tumor-associated antigens; and

[0120] (c) comparing the measured amount of the anti-p53 antibody with a predetermined reference amount of the anti-p53 antibody for the one or more tumor-associated antigens;

[0121] The tumor-associated antigen comprises one or more proteins selected from the group consisting of wild-type p53 isoform A, p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1.

[0122] The isotype of the anti-p53 antibody is at least one selected from the group consisting of IgA antibody, IgM antibody, and IgE antibody.

[0123] The wild-type p53 isoform A of Embodiment 1-3 may be a protein represented by any one of the following (i''') to (iii''').

[0124] A protein represented by any of the following:

[0125] (i''') A protein consisting of the amino acid sequence of SEQ ID NO: 1.

[0126] (ii''') a protein consisting of an amino acid sequence in which one or several amino acids are added, deleted and / or substituted in the amino acid sequence of SEQ ID NO: 1, and an anti-p53 antibody that specifically binds to the protein of SEQ ID NO: 1 has the same level of specific binding as the protein of SEQ ID NO: 1.

[0127] (iii''') a protein consisting of an amino acid sequence having 95% or more homology with any amino acid sequence of SEQ ID NO: 1, and an anti-p53 antibody that specifically binds to the protein of SEQ ID NO: 1 has the same level of specific binding as the protein of SEQ ID NO: 1.

[0128] That is, the method of embodiment 1-3 is the same as that of embodiment 1-1 above, except that at least one of p53 isoforms A to C and the p53 isoform mutants of embodiment 1-1 is used as the tumor-associated antigen, and the isotype of the antibody to be measured is at least one selected from the group consisting of IgA antibody, IgM antibody and IgE antibody. Specifically, the definition of each term, the details of each step, the preferred embodiment, etc. are the same as those of embodiment 1-1 above.

[0129] For example, similarly to the above-mentioned embodiment 1-1, it is preferred to use in combination two or more of the wild-type p53 isoform A, p53 isoform B, p53 isoform C and the p53 isoform mutant of embodiment 1-1 as tumor-associated antigens. Furthermore, for example, the isotype of the anti-p53 antibody is preferably selected from two or more of the group consisting of IgA antibodies, IgM antibodies and IgE antibodies. When there are two or more isotypes of the anti-p53 antibody, it is preferred to perform the above-mentioned steps (b) to (c) on various isotypes of the anti-p53 antibody. Moreover, in the method of embodiment 1-3, when determining two or more isotypes of anti-p53 antibodies, the isotype of the anti-p53 antibody can be a combination of one or more selected from the group consisting of IgA antibodies, IgM antibodies and IgE antibodies and IgG antibodies.

[0130] (Implementation 1-4)

[0131] The method of Embodiments 1 to 4 is a method for collecting data for diagnosis, onset risk assessment, malignancy assessment and / or prognosis prediction of cancer by measuring two or more isotypes of anti-p53 antibodies.

[0132] As described in Embodiment 1-3, according to the knowledge found by the present inventors, it is speculated that the cancer detection result based on the amount of anti-p53 antibody specifically binding to any of the p53 isoform A, p53 isoform B, p53 isoform C, and the p53 isoform mutant of Embodiment 1-1 is different depending on the type of antibody isotype. Therefore, according to the method of Embodiment 1-4 described below, it is possible to widely identify anti-p53 antibodies in samples that cannot be identified by conventional methods. Therefore, data for cancer diagnosis, morbidity risk determination, malignancy determination, and / or prognosis prediction can be collected with good sensitivity while maintaining high specificity.

[0133] The method of embodiments 1-4 is a method for collecting data for cancer diagnosis, risk assessment, malignancy assessment and / or prognosis prediction, comprising:

[0134] (a) a step of inducing an antigen-antibody reaction by contacting a sample from a subject with one or more tumor-associated antigens;

[0135] (b) a step of measuring the amount of the anti-p53 antibody in the sample that specifically binds to the one or more tumor-associated antigens; and

[0136] (c) comparing the measured amount of the anti-p53 antibody with a predetermined reference amount of the anti-p53 antibody for the one or more tumor-associated antigens;

[0137] The tumor-associated antigen comprises one or more proteins selected from the group consisting of wild-type p53 isoform A, p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1.

[0138] The isotype of the anti-p53 antibody is two or more selected from the group consisting of IgG antibody, IgA antibody, IgM antibody, and IgE antibody.

[0139] That is, the method of embodiment 1-4 is the same as that of embodiment 1-1 above, except that at least one of p53 isoforms A to C and the p53 isoform mutants of embodiment 1-1 is used as the tumor-associated antigen, and the isotypes of the antibody to be measured are two or more. Specifically, the definition of each term, the details of each step, the preferred embodiment, etc. are the same as those of embodiment 1-1 above.

[0140] For example, similarly to the above-mentioned embodiment 1-1, it is preferred to use two or more of wild-type p53 isoform A, p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1 in combination as tumor-associated antigens. Furthermore, for example, similarly to the above-mentioned embodiment 1-1, it is preferred to perform the above-mentioned steps (b) to (c) on various isotypes of anti-p53 antibodies. Moreover, in the method of embodiment 1-3, the isotype of the anti-p53 antibody preferably includes IgG antibody and IgA antibody, and more preferably a combination of IgG antibody and IgA antibody.

[0141] <Embodiment 2: Cancer diagnosis kit>

[0142] The cancer diagnosis kit of embodiment 2 includes: a kit related to the above-mentioned p53 isoform mutant with a deletion mutation (embodiment 2-1); a kit related to p53 isoform B, p53 isoform C and the above-mentioned p53 isoform mutant with a deletion mutation (embodiment 2-2); and a kit related to p53 isoform A, p53 isoform B, p53 isoform C and the above-mentioned p53 isoform mutant with a deletion mutation (embodiment 2-3). It should be noted that the cancer diagnosis kit in each embodiment may appropriately contain any reagents, labeled antibodies, etc. known to those skilled in the art for measuring the amount of anti-p53 antibodies as cancer markers. The following is a detailed description.

[0143] (Implementation Method 2-1)

[0144] The cancer diagnosis kit of embodiment 2-1 contains one or more of the p53 isoform mutants of embodiment 1-1. The preferred amino acid sequence of the p53 isoform mutant of embodiment 1-1 contained in the cancer diagnosis kit is the same as the sequence described in embodiment 1-1.

[0145] The cancer diagnosis kit contains, as the tumor-associated antigen, not only the p53 isoform mutant of embodiment 1-1 but also one or more of wild-type p53 isoform A, p53 isoform B, and p53 isoform C. Similarly to the above-mentioned embodiment 1-1, the cancer diagnosis kit preferably contains two or more of the p53 isoform mutants of embodiment 1-1, more preferably contains three or more, further preferably contains four or more, and particularly preferably contains five or more.

[0146] (Implementation method 2-2)

[0147] The cancer diagnosis kit of embodiment 2-2 contains one or more of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1. The amino acid sequence and preferred amino acid sequence of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1 contained in the cancer diagnosis kit are the same as those described in the above-mentioned embodiments 1-1 and 1-2.

[0148] The cancer diagnosis kit of embodiment 2-2, similarly to embodiment 1-2, contains as tumor-associated antigens not only one or more of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1, but may also contain wild-type p53 isoform A. In addition, similarly to embodiment 1-2, the cancer diagnosis kit preferably contains two or more of p53 isoform B, p53 isoform C, and the p53 isoform mutant of embodiment 1-1, more preferably contains three or more, further preferably contains four or more, and particularly preferably contains five or more.

[0149] (Implementation 2-3)

[0150] The cancer diagnosis kit of embodiment 2-3 contains one or more of wild-type p53 isoform A, p53 isoform B, p53 isoform C and the p53 isoform mutant of embodiment 1-1. The amino acid sequence and preferred amino acid sequence of the wild-type p53 isoform A, p53 isoform B, p53 isoform C and the p53 isoform mutant of embodiment 1-1 contained in the cancer diagnosis kit are the same as those described in the above-mentioned embodiments 1-1, 1-2 and 1-3.

[0151] The cancer diagnostic kit of embodiment 2-3, like the above-mentioned embodiments 1-3 and 1-4, preferably comprises two or more of the wild-type p53 isoform A, p53 isoform B, p53 isoform C and the p53 isoform mutant of embodiment 1-1, more preferably comprises three or more, further preferably comprises four or more, and particularly preferably comprises five or more.

[0152] The methods and cancer diagnostic kits according to embodiments 1 to 2 of the present invention can greatly contribute to the diagnosis of cancer, the determination of the risk of developing the disease, the determination of the degree of malignancy, and / or the prediction of the prognosis. Furthermore, the knowledge found by the present inventors regarding the existence of anti-p53 antibodies that have specific binding properties to p53 isoform mutants that cannot be recognized by wild-type p53 isoform A is expected to be relevant to the development of various cancer treatments, antibodies for treatment, vaccines, and the like in the future.

[0153] The outline of the present invention has been described above. The method for collecting data for diagnosing cancer, determining the risk of developing the disease, determining the degree of malignancy, and / or predicting prognosis, and the cancer diagnosis kit according to the present embodiment are summarized below.

[0154] The method of the first aspect of the present invention is a method for collecting data for cancer diagnosis, risk assessment, malignancy assessment and / or prognosis prediction, comprising:

[0155] (a) a step of inducing an antigen-antibody reaction by contacting a sample from a subject with one or more tumor-associated antigens;

[0156] (b) a step of measuring the amount of the anti-p53 antibody in the sample that specifically binds to the one or more tumor-associated antigens; and

[0157] (c) comparing the measured amount of the anti-p53 antibody with a predetermined reference amount of the anti-p53 antibody for the one or more tumor-associated antigens;

[0158] The above-mentioned tumor-associated antigen includes one or more p53 isoform mutants consisting of an amino acid sequence having a mutation in any one of the amino acid sequences of SEQ ID NOs: 1 to 3, and the above-mentioned mutation includes any one of an N-terminal deletion mutation, a C-terminal deletion mutation, and a deletion mutation of both the N-terminus and the C-terminus.

[0159] In the above method, the p53 isoform mutant is preferably a protein represented by any one of the following:

[0160] (i) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 35;

[0161] (ii) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of SEQ ID NOs: 4 to 35, and the anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above has the same level of specific binding activity as the protein of the corresponding sequence number; and

[0162] (iii) a protein consisting of an amino acid sequence having 95% or more homology to any one of the amino acid sequences of SEQ ID NOs: 4 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above and has the same level of specific binding as the protein of the corresponding sequence number.

[0163] In the above method, the p53 isoform mutant is more preferably a protein represented by any one of the following:

[0164] (I) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NOs: 16 to 24, SEQ ID NO: 26, and SEQ ID NOs: 29 to 35;

[0165] (II) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16 to SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 29 to SEQ ID NO: 35, and the anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) above has the same level of specific binding activity as the protein of the corresponding sequence number; and

[0166] (III) a protein consisting of an amino acid sequence having 95% or more identity to any one of SEQ ID NOs: 4 to 6, 7, 8, 10, 11, 14, 16 to 24, 26, and 29 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) above, having the same level of specific binding activity as the protein of the corresponding sequence number.

[0167] In the above method, the tumor-associated antigen preferably further comprises one or more p53 isoforms consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

[0168] In the above method, the isotype of the anti-p53 antibody is one or more selected from the group consisting of IgG antibody, IgA antibody, IgM antibody and IgE antibody. When the isotype of the anti-p53 antibody is two or more, it is particularly preferred to perform the above steps (b) to (c) on various isotypes of the anti-p53 antibody.

[0169] The cancer diagnostic kit of the second aspect of the present invention comprises one or more p53 isoform mutants consisting of an amino acid sequence having a mutation in any one of the amino acid sequences of SEQ ID NOs: 1 to 3, and the above mutation comprises any one of an N-terminal deletion mutation, a C-terminal deletion mutation, and a deletion mutation of both the N-terminus and the C-terminus.

[0170] In the above-mentioned cancer diagnosis kit, the above-mentioned p53 isoform mutant is preferably a protein represented by any one of the following:

[0171] (i) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 35;

[0172] (ii) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of SEQ ID NOs: 4 to 35, and the anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above has the same level of specific binding activity as the protein of the corresponding sequence number; and

[0173] (iii) a protein consisting of an amino acid sequence having 95% or more homology to any one of the amino acid sequences of SEQ ID NOs: 4 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above and has the same level of specific binding as the protein of the corresponding sequence number.

[0174] In the above-mentioned cancer diagnosis kit, the above-mentioned p53 isoform mutant is more preferably a protein represented by any one of the following:

[0175] (I) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NOs: 16 to 24, SEQ ID NO: 26, and SEQ ID NOs: 29 to 35;

[0176] (II) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16 to SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 29 to SEQ ID NO: 35, and the anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) above has the same level of specific binding activity as the protein of the corresponding sequence number; and

[0177] (III) a protein consisting of an amino acid sequence having 95% or more identity to any one of SEQ ID NOs: 4 to 6, 7, 8, 10, 11, 14, 16 to 24, 26, and 29 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) above, having the same level of specific binding activity as the protein of the corresponding sequence number.

[0178] Example

[0179] The present invention is further described in detail below by way of examples, but the present invention is not limited by the examples.

[0180] In this example, it was investigated whether there were anti-p53 antibodies specific to each p53 isoform mutant having any one of the N-terminal deletion mutation, the C-terminal deletion mutation, and the deletion mutation of both the N-terminus and the C-terminus, p53 isoform B, or p53 isoform C. Furthermore, it was also investigated whether there was a difference in the detection results between IgG antibodies and IgA antibodies.

[0181] Specifically, the anti-p53 antibodies contained in the sample were profiled by using a protein microarray detection of wild-type p53 isoform A, p53 isoform B, p53 isoform C, and synthetic human proteins of each p53 isoform mutant using IgG antibodies or IgA antibodies. As samples, human plasma from 60 individuals (samples No. 1 to No. 60) was used. The specific method is described in detail below.

[0182] <Example 1: Spectrum analysis of IgG antibodies specific to p53 isoform mutants, p53 isoform B, or p53 isoform C>

[0183] In Example 1, 60 human plasma samples were investigated for positive samples in which the IgG antibody of anti-p53 antibody was detected at a standard amount or more using each p53 isoform mutant, p53 isoform B or p53 isoform C. As a control (comparative example), positive samples in which the IgG antibody of anti-p53 antibody was detected at a standard amount or more using wild-type p53 isoform A were investigated.

[0184] (Experimental Method)

[0185] The protein microarray used a microarray of 16,680 synthetic human proteins including wild-type p53 isoform A, p53 isoform B, p53 isoform C, and mutants of each p53 isoform. The protein microarray was produced by the Medical-Industrial Translational Research Center of Fukushima Medical University.

[0186] Wild-type p53 isoform A, p53 isoform B, and p53 isoform C are proteins composed of the amino acid sequences of sequence numbers 1 to 3, respectively. As p53 isoform mutants, p53 isoforms (p53 isoforms G, I, H, D, E, F, J, K, L) having N-terminal deletion mutations, p53 isoforms having only C-terminal deletion mutations caused by nonsense mutations, and p53 isoforms having deletion mutations of both N-terminal deletion mutations and C-terminal deletion mutations are printed. Specifically, various p53 isoform mutants composed of amino acid sequences of sequence numbers 4 to 35 having any of the N-terminal deletion mutations, C-terminal deletion mutations, and deletion mutations of both N-terminal and C-terminal deletion mutations in the amino acid sequences of sequence numbers 1 to 3 are printed as shown in Tables 2 and 3 below.

[0187] The protein microarray used was a protein microarray prepared by the method described in Naoki Goshima, Yoshifumi Kawamura, Akiko Fukumoto et al., "Human protein factory for converting the transcriptome into an invitro-expressed proteome.", Nature methods, 12, 1011-1027, 2008. Specifically, as the protein microarray, an array prepared by printing synthetic human proteins containing wild-type p53 isoform A, p53 isoform B, p53 isoform C and various p53 isoform mutants on a slide glass (manufactured by Matsunami Glass Industries, Ltd.) using an ultra-micro dispensing device (manufactured by Microdevice Co., Ltd.) according to the protocol recommended by the company was used.

[0188] After the prepared protein microarray was immersed in ethanol for 30 seconds, the protein microarray was washed twice with ultrapure water for 30 seconds each time. Then, the protein microarray was immersed in a protein microarray dedicated solution A (manufactured by Fukushima Projection Phactory Co., Ltd.) and shaken for 5 minutes at 26°C. Then, the protein microarray was immersed in BlockingOne (manufactured by Nakalai Test Co., Ltd.) and shaken for 1 hour at 26°C, and the solution was replaced with a protein microarray dedicated solution A (manufactured by Fukushima Projection Phactory Co., Ltd.).

[0189] Then, add 1 / 4000 of the goat reference antibody mixture I (made by Fukushima Projectile Phaktori Co., Ltd.) to the protein microarray-specific primary antibody diluent (made by Fukushima Projectile Phaktori Co., Ltd.). Furthermore, use this solution to dilute the human plasma of 60 people to a concentration of 1 / 2000, and prepare each primary antibody solution. Add 2mL of each prepared primary antibody solution to the protein microarray box P-DE1 (made by Fukushima Projectile Phaktori Co., Ltd.). Then insert the protein microarray replaced with solution A (made by Fukushima Projectile Phaktori Co., Ltd.) into the box and cover it. Then, the protein microarray covered with the lid is sealed with 8 special clips and reacted at 37°C for 17 hours (primary antibody reaction).

[0190] After the primary antibody reaction, the protein microarray was washed three times for 1 minute each time using a protein microarray solution A (manufactured by Fukushima Projectile Co., Ltd.) at 26° C. The washed protein microarray was then left to stand for 5 minutes and washed again three times for 3 minutes each time.

[0191] Then, add 1 / 500 of Cy3 labeled anti-goat IgG antibody and Alexa Fluor647 (registered trademark) labeled anti-human IgG antibody to the protein microarray dedicated secondary antibody diluent (manufactured by Fukushima Projectile Co., Ltd.) to prepare the secondary antibody solution. 2 mL of the prepared secondary antibody solution is added to the protein microarray cassette P-DE1 (manufactured by Fukushima Projectile Co., Ltd.). Then, the cleaned protein microarray is inserted into the cassette and the lid is closed. Next, the protein microarray covered with the lid is sealed with 8 special clips and reacted at 26°C for 1 hour (secondary antibody reaction).

[0192] After the secondary antibody reaction, the protein microarray was washed three times for 1 minute each time with protein microarray solution A (manufactured by Fukushima Projectile Co., Ltd.) at 26° C. Then, the washed protein microarray was left to stand for 5 minutes and washed again three times for 3 minutes each time.

[0193] Next, the protein microarray was washed twice with protein microarray solution B (manufactured by Fukushima Projection Filter Co., Ltd.) for 30 seconds each time, and then washed four times with protein microarray final washing solution (manufactured by Fukushima Projection Filter Co., Ltd.) for 30 seconds each time. The washed protein microarray was centrifuged at 1500 rpm for 1 minute and dried. The protein microarray was then scanned with a GenePix 4000B scanner.

[0194] The standardization of the numerical values ​​between each sample is performed using Excel software (Microsoft, Bellevue, WA, USA) according to the following method. First, the numerical values ​​of the fluorescence intensity of Cy3 from the reference antibody are calculated to be several times the average value of all the measured values ​​of the fluorescence intensity of Cy3. Then, the fluorescence intensity of AlexaFluor (registered trademark) 647 of the spots of each protein of each sample measured by the scanner is divided by this value. At this time, when the numerical value of the fluorescence intensity of Cy3 from the reference antibody is zero, the data of the spot is set to a poor spot and is not adopted. Then, the measured value of the spot without adding human plasma is subtracted from the division value of the fluorescence intensity of the spots of the above-mentioned each protein to remove the background. Then, after subtracting the 25th percentile value of each sample from the calculated value, the value is divided by the SD value. Then, the calculated value is divided by the 75th percentile value of each sample, and the value is multiplied by 10.

[0195] When the standardized value exceeds 50 in this way, it is considered that the IgG antibody (IgA antibody in Example 2 described later) of the anti-p53 antibody specific to the target protein is measured to be above the reference amount. That is, based on the determination of the target protein, the sample can be determined to be positive. On the other hand, when the standardized value is below 50, it is considered that the IgG antibody (IgA antibody in Example 2 described later) of the anti-p53 antibody specific to the target protein is measured to be less than the reference amount. That is, based on the determination of the target protein, the sample can be determined to be negative. It should be noted that several proteins in the wild-type p53 isoform A, p53 isoform B, p53 isoform C and each p53 isoform mutant as the target protein are measured simultaneously in multiple spots on a slide. In this case, when more than half of the multiple spots are determined to be positive, it is considered to be positive.

[0196] (Results and Investigation)

[0197] Table 2 below shows the details of the structures of the amino acid sequences of the proteins of wild-type p53 isoform A, p53 isoform B, p53 isoform C, and each p53 isoform mutant printed on the array and the details of the measurement results of IgG antibodies of anti-p53 antibodies against these proteins.

[0198]

[0199] As shown in Table 2, in the measurement of the IgG antibody of the anti-p53 antibody of the wild-type isoform A of the comparative example, 4 samples were judged to be positive among the 60 samples.

[0200] On the other hand, in the determination of IgG antibodies of anti-p53 antibodies of each p53 isoform mutant of Examples 1-1 to 1-32, only Examples 1-11, 1-12, and 1-13 obtained completely the same results as those of the Comparative Examples. In particular, in the determination of IgG antibodies of anti-p53 antibodies of each p53 isoform mutant of Examples 1-1, 1-2, 1-4, 1-5, 1-7, 1-8, 1-14 to 1-17, 1-20, 1-21, 1-23, and 1-29 to 1-32, samples that were determined to be negative according to the Comparative Examples were determined to be positive.

[0201] From the results shown in Table 2, it is clear that there are a variety of anti-p53 IgG antibodies that specifically bind to each p53 isoform mutant and cannot be recognized by the wild-type p53 isoform A.

[0202] Although not shown in Table 2 above, no significant difference was observed in the measurement results of IgG antibodies of anti-p53 antibodies obtained using p53 mutants having only amino acid substitutions caused by missense mutations in the amino acid sequence shown in SEQ ID NO: 1, compared with the comparative example. Therefore, it is known that such specific anti-p53 antibodies are significantly present in p53 isoform mutants having N-terminal deletion mutations, C-terminal deletion mutations, or deletion mutations at both N-termini and C-termini.

[0203] In addition, there are p53 isoform mutants in which there are no positive samples in Examples 1-1 to 1-32. However, based on the various detection results of the positive samples in Example 1, it is speculated that by measuring with other samples, IgG antibodies of anti-p53 antibodies that specifically bind to each p53 isoform mutant can be found.

[0204] These results show that by using one or more p53 isoform mutants of Examples 1-1 to 1-32 in combination, anti-p53 antibodies that were not previously identifiable can be detected. By combining the detection results, it is sufficient to speculate that when collecting data for cancer diagnosis, etc., it is possible to maintain high specificity while improving sensitivity.

[0205] Furthermore, as shown in Table 2 above, in the measurement of the IgG antibody of the anti-p53 antibody against p53 isoform C in Reference Example 1-2, the sample determined to be negative in the Comparative Example was determined to be positive.

[0206] From the results of Reference Example 1-2 shown in Table 2 above, it is known that there are anti-p53 IgG antibodies that specifically bind to p53 isoform C and cannot be recognized by wild-type isoform A.

[0207] It should be noted that the measurement results of p53 isoform B in Reference Example 1-1 were the same as those in Comparative Example (WT). However, based on the various detection results of the positive samples in Reference Example 1-2 and Examples 1-1 to 1-32, it can be inferred that IgG antibodies of anti-p53 antibodies that specifically bind to p53 isoform B can be found by measuring with other samples.

[0208] These results show that by using one or more p53 isoforms B, p53 isoforms C, and the p53 isoform mutants of Examples 1-1 to 1-32 in combination, anti-p53 antibodies that were not previously identifiable can be detected. As a result, it is possible to predict that when collecting data for cancer diagnosis, etc., it is possible to maintain high specificity while improving sensitivity.

[0209] <Example 2: Spectrum analysis of IgA antibodies specific to p53 isoform mutants or p53 isoforms A to C>

[0210] In Example 2, 60 human plasma samples were examined for positive samples in which IgA antibodies against p53 antibodies were detected at a reference level or above using each p53 isoform mutant or p53 isoforms A to C. As controls (comparative examples), similar to Example 1, positive samples in which IgG antibodies against p53 antibodies were detected at a reference level or above using wild-type p53 isoform A were used.

[0211] (Experimental Method)

[0212] A primary antibody solution was prepared by adding a 4000th amount of goat reference antibody mixture II and a pre-reacted antibody of Cy3-labeled anti-goat IgG antibody to the primary antibody dilution for protein microarray, and a secondary antibody solution was prepared by adding a 500th amount of DyLight650-labeled anti-human IgA antibody to the secondary antibody dilution for protein microarray. Otherwise, the IgA antibody of the anti-p53 antibody against each p53 isoform mutant and p53 isoforms A to C was measured, standardized, and positive or negative determined by the same method as Example 1.

[0213] (Results and Investigation)

[0214] Table 3 below shows the details of the structures of the amino acid sequences of the proteins of wild-type p53 isoform A, p53 isoform B, p53 isoform C, and each p53 isoform mutant printed on the array and the details of the measurement results of IgG antibodies or IgA antibodies of anti-p53 antibodies against these proteins.

[0215]

[0216] As described above, in the determination of IgG antibodies of anti-p53 antibodies of wild-type isoform A in the comparative example, 4 samples were judged to be positive among 60 samples. On the other hand, as shown in the above Table 3, in the determination of IgA antibodies of anti-p53 antibodies of each p53 isoform mutant of Examples 2-1 to 2-32, there was no example with the same result as the comparative example. In particular, in the determination of IgA antibodies of anti-p53 antibodies of each p53 isoform mutant of Examples 2-1 to 2-3, 2-11, 2-13 to 2-19, 2-21, 2-26 to 2-30 and 2-32, samples judged to be negative according to the comparative example were judged to be positive.

[0217] From the results shown in Table 3 above, it can be seen that there are not only IgG antibodies but also a variety of IgA antibodies that specifically bind to each p53 isoform mutant. Furthermore, by comparing Table 1 of Example 1 with Table 2 above, it can be seen that even when the same p53 isoform mutant is used, the detection results of positive samples obtained using IgG antibodies and the detection results of positive samples obtained using IgA antibodies are not necessarily consistent.

[0218] In addition, there are p53 isoform mutants with zero positive samples in Examples 2-1 to 2-32. However, based on the various detection results of the positive samples in Example 2, it is speculated that IgA antibodies of anti-p53 antibodies that specifically bind to each p53 isoform mutant can be found by measuring other samples.

[0219] From these results, it is clear that by using one or more p53 isoform mutants of Examples 2-1 to 2-32 (p53 isoform mutants represented by the amino acid sequences of SEQ ID NOs: 4 to 35) in combination to measure both IgG antibodies and IgA antibodies, it is possible to detect more widely anti-p53 antibodies that were not previously identifiable. It is also sufficient to speculate that by combining the results, when collecting data for cancer diagnosis, etc., the sensitivity can be further improved while maintaining high specificity.

[0220] Furthermore, as shown in Table 3, there was no reference example in Reference Examples 2-1 to 2-3 that had the same results as the Comparative Examples. In particular, in the determination of IgA antibodies of the anti-p53 antibody of the wild-type p53 isoform A of Reference Example 2-1 and the p53 isoform C of Reference Example 2-3, a total of 3 samples that were determined to be negative according to the Comparative Examples were determined to be positive.

[0221] From the results shown in Table 3 above, it can be seen that there are not only IgG antibodies but also a variety of IgA antibodies that are anti-p53 antibodies that specifically bind to p53 isoforms A to C. Furthermore, by comparing the comparative examples and reference examples in Table 2 of Example 1 with the reference examples in Table 3 above, it can be seen that even when the same p53 isoforms A to C are used, the detection results of positive samples obtained using IgG antibodies and the detection results of positive samples obtained using IgA antibodies are not necessarily consistent.

[0222] In Reference Example 2-2, no positive samples other than the positive samples determined to be positive in the Comparative Example were detected. However, based on the various detection results of the positive samples in Reference Examples 2-1 and 2-3 and Examples 2-1 to 2-32, it is speculated that IgA antibodies to anti-p53 antibodies that specifically bind to p53 isoform B can be found by measuring with other samples.

[0223] From these results, it can be seen that by using p53 isoforms A to C and one or more of the p53 isoform mutants of Examples 2-1 to 2-32 in combination to measure both IgG antibodies and IgA antibodies, it is possible to detect more widely anti-p53 antibodies that were not previously recognized. As a result, it is sufficient to speculate that when collecting data for cancer diagnosis, etc., the sensitivity can be further improved while maintaining high specificity.

[0224] For example, the detection results of positive samples obtained using the p53 isoform mutants of Examples 1-1 to 1-32 and Examples 2-1 to 2-32 related to the method of Embodiment 1-1 are shown in Table 4 below.

[0225]

[0226] As shown in Table 4, when both IgG and IgA antibodies of anti-p53 antibodies were measured using all p53 isoform mutants shown in the amino acid sequences of SEQ ID NOs. 4 to 35, 23 positive samples were detected among 60 samples. On the other hand, according to the conventional method of measuring IgG antibodies of anti-p53 antibodies using wild-type isoform A, only 4 positive samples were detected. That is, it is estimated that there are many samples that are actually positive but are judged to be negative in the case of the conventional method.

[0227] Therefore, according to the results of Examples 1 and 2, by using one or more of the p53 isoforms A to C and p53 isoform mutants represented by the amino acid sequences of SEQ ID NOs: 1 to 35 in combination to measure both IgG antibodies and IgA antibodies, it is possible to detect a wider range of anti-p53 antibodies that have not been recognized in the past. As a result, it is estimated that when collecting data for cancer diagnosis, etc., the sensitivity can be significantly improved while maintaining high specificity.

[0228] This application is based on Japanese patent application No. 2022-144861 filed on September 12, 2022, and the contents are included in this application.

[0229] In order to describe the present invention, the present invention is appropriately and fully described above by means of embodiments and examples with reference to specific examples, but it should be recognized that those skilled in the art can easily make changes and / or improvements to the above-mentioned embodiments and examples. Therefore, as long as the changes or improvements implemented by those skilled in the art do not deviate from the level of the protection scope of the claims recorded in the claims, the changes or improvements should be interpreted as being included in the protection scope of the claims.

[0230] Industrial Applicability

[0231] The method and cancer diagnostic kit of the present invention can greatly contribute to the diagnosis of cancer, the determination of the risk of onset, the determination of the degree of malignancy and / or the prediction of prognosis. Furthermore, the knowledge found by the present inventors regarding the existence of anti-p53 antibodies that have specific binding properties to p53 isoform mutants that cannot be recognized by wild-type p53 isoform A is expected to be relevant to the development of various cancer treatments, antibodies for treatment, vaccines, etc. in the future.

Claims

1. A method for collecting data for cancer diagnosis, risk assessment, malignancy assessment and / or prognosis prediction, comprising: (a) a step of inducing an antigen-antibody reaction by contacting a sample from a subject with one or more tumor-associated antigens; (b) a step of measuring the amount of the anti-p53 antibody in the sample that specifically binds to the one or more tumor-associated antigens; and (c) comparing the measured amount of the anti-p53 antibody with a predetermined reference amount of the anti-p53 antibody for the one or more tumor-associated antigens, The tumor-associated antigen comprises one or more p53 isoform mutants consisting of an amino acid sequence having a mutation in any one of the amino acid sequences in SEQ ID NOs: 1 to 3, and the mutation comprises any one of an N-terminal deletion mutation, a C-terminal deletion mutation, and a deletion mutation at both the N-terminus and the C-terminus.

2. The method according to claim 1, wherein: The p53 isoform mutant is a protein represented by any one of the following (i) to (iii): (i) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 35; (ii) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of SEQ ID NOs: 4 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above has the same level of specific binding as the protein of the corresponding sequence number; as well as (iii) a protein consisting of an amino acid sequence having 95% or more homology to any one of the amino acid sequences of SEQ ID NOs: 4 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above, having the same level of specific binding as the protein of the corresponding sequence number.

3. The method according to claim 1, wherein: The p53 isoform mutant is a protein represented by any one of the following (I) to (III): (I) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NOs: 16 to 24, SEQ ID NO: 26, and SEQ ID NOs: 29 to 35; (II) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16 to SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 29 to SEQ ID NO: 35, and the anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) has the same level of specific binding activity as the protein of the corresponding sequence number; and (III) a protein consisting of an amino acid sequence having 95% or more identity to any one of SEQ ID NOs: 4 to 6, 7, 8, 10, 11, 14, 16 to 24, 26, and 29 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) above, having the same level of specific binding as the protein of the corresponding sequence number.

4. The method according to any one of claims 1 to 3, wherein: The tumor-associated antigen further includes one or more p53 isoforms consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 3.

5. The method according to any one of claims 1 to 3, wherein: The isotype of the anti-p53 antibody is one or more selected from the group consisting of IgG antibody, IgA antibody, IgM antibody and IgE antibody, When the anti-p53 antibody has two or more isotypes, steps (b) to (c) are performed on each isotype of the anti-p53 antibody.

6. A cancer diagnosis kit comprising one or more p53 isoform mutants consisting of an amino acid sequence having a mutation in any one of the amino acid sequences of SEQ ID NOs: 1 to 3, wherein the mutation comprises any one of an N-terminal deletion mutation, a C-terminal deletion mutation, and a deletion mutation at both the N-terminus and the C-terminus.

7. The cancer diagnosis kit according to claim 6, wherein: The p53 isoform mutant is a protein represented by any one of the following (i) to (iii): (i) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 35; (ii) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of SEQ ID NOs: 4 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above has the same level of specific binding as the protein of the corresponding sequence number; as well as (iii) a protein consisting of an amino acid sequence having 95% or more homology to any one of the amino acid sequences of SEQ ID NOs: 4 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (i) above, having the same level of specific binding as the protein of the corresponding sequence number.

8. The cancer diagnosis kit according to claim 6 or 7, wherein The p53 isoform mutant is a protein represented by any one of the following (I) to (III): (I) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NOs: 16 to 24, SEQ ID NO: 26, and SEQ ID NOs: 29 to 35; (II) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in any one of the amino acid sequences of SEQ ID NOs: 4 to 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16 to SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 29 to SEQ ID NO: 35, and the anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) has the same level of specific binding activity as the protein of the corresponding sequence number; and (III) a protein consisting of an amino acid sequence having 95% or more identity to any one of SEQ ID NOs: 4 to 6, 7, 8, 10, 11, 14, 16 to 24, 26, and 29 to 35, and an anti-p53 antibody that specifically binds to the protein of the corresponding sequence number described in (I) above, having the same level of specific binding as the protein of the corresponding sequence number.

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