JP2006242869A - Anti-nasp antibody and anti-tmf antibody as tumor marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel tumor marker containing an anti-NASP antibody and/or an anti-TMF antibody, and to provide a novel tumor diagnostic composition containing antigens reacting with the antibodies, a novel tumor diagnostic kit, an antibody detecting method, and a diagnostic method for a tumor. <P>SOLUTION: This tumor marker of the present invention contains the anti-NASP antibody and/or the anti-TMF antibody. The tumor diagnostic composition and the tumor diagnostic kit of the present invention include a NASP and/or a TMF. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a tumor marker, a tumor diagnostic composition, and a tumor diagnostic kit.

  The number of deaths per year due to renal cancer in Japan has been increasing year by year. Among malignant tumors in the urological field, men are the third largest after prostate cancer and bladder cancer, and women are second only to bladder cancer. 2nd place. About 90% of kidney tumors have renal cell carcinoma, and about 3,000 people die each year in Japan. In particular, for patients with stage IV renal cell carcinoma (RCC), there is no improvement in prognosis even with various treatments such as surgical resection, chemotherapy, radiation therapy, and cytokine therapy, and the 2-year survival rate Is about 20% or less.

  In recent years, it has been reported that GM-CSF gene-introduced autologous tumor cell vaccines introduced ex vivo induce anti-tumor immune responses in pre-clinical studies using various mouse tumor models and in human clinical studies. Since then, clinical studies have been conducted in melanoma, RCC, prostate cancer, pancreatic cancer, and non-small cell lung cancer.

  Against this background, clinical research on immune gene therapy targeting RCC patients has been carried out (Non-patent Documents 3 and 4).

  As a result, safety, which is the purpose of Phase I clinical research, was confirmed, and various immunological tests (DTH (delayed skin reaction) of 4 patients (1 to 4 cases) who were treated The results of the test and peripheral blood lymphocyte reaction suggested the immunological efficacy of the vaccine.

  In order to clarify the reason for the patient who showed a good clinical course, detailed immunological examination was conducted.

  First, cellular immunity tests showed oligoclonal proliferation and cytotoxic activity of T lymphocytes after vaccination.

Next, a humoral immunity test was performed. That is, when the serum samples of autologous tumor cells and normal kidney tissue cells were reacted with patient sera before and after the above immunogene therapy (before and after tumor vaccination), the second and second In the 3rd and 4th cases, a signal suggesting induction of humoral immunity against some proteins was detected when the patient serum after treatment was reacted (Non-patent Document 4).
Childs, R., et al., N. Engl. J. Med., 2000. 343 (11): p. 750-8 Dranoff, G., Immunol. Rev., 2002. 188: p. 147-54 Tani, K., et al., Cancer Chemother Pharmacol, 2000. 46 Suppl: p. S73-6 Tani, K., et al., Mol. Ther., 2004. 10 (4): p. 799-816

  However, it was not clear what kind of signal suggested the induction of humoral immunity, that is, what kind of antibody was produced by the induction.

  Therefore, the problem to be solved by the present invention is to identify (identify) the antibody produced by the induction of humoral immunity, to provide a novel tumor marker containing the antibody, and to react with the antibody. It is an object of the present invention to provide a novel tumor diagnostic composition containing a possible antigen, a tumor diagnostic kit, a detection method for the antibody, and a tumor diagnostic method.

  The present inventor has intensively studied to solve the above problems. In the process, a cDNA expression phage library derived from stage IV renal cell carcinoma (RCC) cells is prepared and reacted with the patient serum (ie, serum IgG) after the aforementioned immunogene therapy (after tumor vaccination). The “SEREX method”, which is a method for identifying antigens that strongly induced humoral immunity in the patient, was used to identify tumor-specific antigens (described in detail later).

  As a result, the present inventor succeeded in identifying a novel antigen useful as a tumor marker, and found that the antigen is novel and useful as a tumor marker in various tumors other than RCC. The present invention has been completed based on the above findings.

  That is, the present invention is as follows.

 (1) A tumor marker comprising an anti-NASP antibody and / or an anti-TMF antibody.

 (2) A tumor diagnostic composition comprising NASP and / or TMF.

 (3) A tumor diagnostic kit comprising NASP and / or TMF.

 (4) A method for detecting anti-NASP antibody and / or anti-TMF antibody, comprising reacting NASP and / or TMF with a biological sample.

 (5) A method for diagnosing a tumor, wherein the detection result obtained by the method (4) is used as an index.

  In the above (1), (2), (3) and (5), for example, the tumor is from the group consisting of renal cell cancer, colon cancer, melanoma, esophageal cancer, lung cancer, endometrial cancer, and cervical cancer. It is preferable that at least one selected.

  According to the present invention, a novel and extremely useful tumor marker for various tumors, a diagnostic composition for various tumors, a diagnostic kit, an antibody detection method used as a tumor marker, and a diagnosis for various tumors A method can be provided.

Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these descriptions, and modifications other than the following exemplifications can be made as appropriate without departing from the spirit of the present invention.
1. Overview The prognosis for patients with stage IV renal cell carcinoma (RCC) is extremely poor, and no improvement has been seen using treatment such as surgical resection, chemotherapy, radiation therapy, and cytokine therapy. A recently conducted clinical study of gene therapy for RCC patients was conducted by introducing / expressing the GM-CSF gene into autologous tumor cells and inactivating it by irradiation, and then administering it as a tumor vaccine to patients. -Anti-tumor immunity induced in the patient's body was expected by artificially increasing the original immunogenicity of the tumor through the action of -CSF. As described above, induction of cell-induced T cell activity against autologous RCC and induction of a humoral immune response have already been reported in patients who have already undergone the clinical study.

  The present inventor, for the purpose of specifying the antibody produced by induction of this humoral immune response (which can be used as a tumor marker) Using the “SEREX method”, several antibodies (antibodies capable of reacting with the SEREX antigen) were successfully identified. Therefore, their RCC specificity was examined from the viewpoints of the expression level of antigen mRNA in normal tissues and cancer tissues, and the appearance frequency of serum antibodies in healthy people and cancer patients.

  As a result, the expression of tNASP (testicular nuclear autoantigenic sperm protein) mRNA tended to be significantly increased in cancer tissues. Moreover, the tendency which the antibody titer with respect to tNASP and TMF (TATA element modulatory factor 1) was increasing significantly after GM-CSF gene introduction vaccination was recognized. In particular, the anti-TMF antibody titer was RCC (69% positive for cancer-bearing patients, compared to the detection limit for all healthy individuals at the stage of examining sera from 23 healthy individuals and a total of 107 patients with various cancers. Rate) and more than 50% of patient sera were positive in colorectal cancer and uterine cancer. The knowledge based on the above research results will greatly suggest future diagnosis and treatment research and development of various cancers.

In addition, it is known that human NASP splicing variants include variants 1, 2 and 3. In this specification, variants 1 and 2 are tNASP and variant 3 is sNASP (somatic nuclear autoantigenic sperm protein ), Which means something different from tNASP and sNASP, which are generally known as NASP variants of mice.

2. Types of tumors The types of tumors to which the present invention is applied are not particularly limited, and examples thereof include the following tumors.

(a) Oral, nasal and pharyngeal tumors: tongue cancer, gingival cancer, malignant lymphoma, malignant melanoma, maxillary cancer, nasal cancer, nasal cavity cancer, laryngeal cancer, pharyngeal cancer, etc.
(b) Cranial nervous system tumors: glioma, meningioma, etc.
(c) Thyroid tumors: papillary adenocarcinoma, follicular thyroid cancer, medullary thyroid cancer, etc.
(d) Respiratory tumors: lung cancer (squamous cell carcinoma, adenocarcinoma, alveolar cell carcinoma, large cell undifferentiated cancer, small cell undifferentiated cancer, and carcinoid), etc.
(e) Breast tumors: breast cancer, breast paget disease, breast sarcoma, etc.
(f) Blood tumors: Acute myeloid leukemia, acute promedullary leukemia, acute myeloid monocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia, chronic myelogenous leukemia, chronic lymphocytic Leukemia, adult T-cell leukemia, malignant lymphoma (lymphosarcoma, reticulosarcoma, and Hodgkin's disease), multiple myeloma, and primary macroglobulinemia
(g) Gastrointestinal tumors: esophageal cancer, stomach cancer, gastric / intestinal malignant lymphoma, pancreatic cancer, biliary tract cancer, gallbladder cancer, duodenal cancer, colon cancer, liver cancer, etc.
(h) Tumors of female genitals: uterine cancer (such as endometrial cancer and cervical cancer), ovarian cancer, and uterine sarcoma (leiomyosarcoma, rhabdomyosarcoma, lymphosarcoma, and reticulosarcoma), etc.
(i) Urinary tumors include urinary malignant tumors (prostate cancer, renal cancer (renal cell carcinoma, etc.), bladder cancer, testicular tumor, and urethral cancer)
(j) Examples of musculoskeletal tumors include rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, synovial sarcoma, myxosarcoma, liposarcoma, Ewing sarcoma, and multiple myeloma
(k) Skin tumors include skin cancer, cutaneous Bowen's disease, cutaneous Paget's disease, and cutaneous malignant melanoma (melanoma). In addition, one or more of the above tumors may be applied. May be combined.

Of those listed above, the tumor to which the present invention is applied is preferably at least one selected from the group consisting of renal cell carcinoma, colon cancer, melanoma, esophageal cancer, lung cancer, endometrial cancer, and cervical cancer. It is a seed. In particular, when an anti-tNASP antibody is used as a tumor marker (clinical clinical marker), colon cancer, renal cell cancer, esophageal cancer, and endometrial cancer are preferable, and endometrial cancer is more preferable. Similarly, when using an anti-TMF antibody, colon cancer, melanoma, renal cell cancer, esophageal cancer, lung cancer, uterine body cancer, and cervical cancer are preferable, and more preferably, colon cancer, melanoma, renal cell cancer, Esophageal cancer, endometrial cancer, and cervical cancer, more preferably colon cancer, melanoma, renal cell cancer, endometrial cancer, and cervical cancer.

3. SEREX method The anti-NASP antibody (anti-tNASP antibody and anti-sNASP antibody) and anti-TMF antibody used as tumor markers of the present invention are both SEREX by using the SEREX method (Serological identification by recombinant expression cloning method). It has been identified (identified) as being capable of reacting with an antigen.

  For the SEREX method, see `` FIG. 1 '' and `` Kiniwa, Y., et al., Tumor antigens isolated from a patient with vitiligo and T-cell-infiltrated melanoma. Cancer Res., 2001. 61 (21): p. 7900-7 "describes its schematic diagram and details. The outline is as follows.

That is, an expression library is constructed using cancer cell mRNA and a λ phage expression vector. The λ phage infected with E. coli (XL-1blue MRF ′) is cultured on NZY plate medium to make a lysis plaque. By covering the plate with a nitrocellulose membrane infiltrated with IPTG, a protein encoded by a cDNA derived from a cancer cell incorporated into λ phage was produced in the plaque, and this protein was transferred onto the nitrocellulose membrane. From serum (eg, serum of patients who had a good clinical course after gene therapy (after tumor vaccination)) against E. coli and λ phage using a column packed with lysates of E. coli and λ phage and Sepharose 4B After removing the antibody, it is diluted as appropriate (for example, about 100 to 800 times) to prepare a serum sample. The blocked nitrocellulose membrane is washed and reacted with the prepared serum sample. Further washing is performed, and an enzyme-labeled mouse anti-human IgG monoclonal antibody is reacted as a secondary antibody. After further washing, the nitrocellulose membrane is reacted with a chromogenic substrate, the plaque color development status is confirmed, and positive plaque is picked up. Thereafter, using the phage recovered from the positive plaque, the same operation as described above is carried out, and another thing that can be judged as a positive plaque is picked up again. The insert DNA portion is amplified by PCR, and only those whose band is confirmed at the same position by electrophoresis are identified as final positive plaques by DNA sequence analysis and used as SEREX antigen.

4). Tumor marker As described above, the tumor marker of the present invention comprises an anti-NASP antibody (anti-tNASP antibody and anti-sNASP antibody) and / or an anti-TMF antibody, ie, an anti-NASP antibody and / or An anti-TMF antibody is used as a tumor marker (clinical marker for cancer).

Here, the tumor marker of the present invention may require either an anti-NASP antibody or an anti-TMF antibody, for example, for the purpose of detecting two or more tumors, etc. Not done. When an anti-NASP antibody is essential, either an anti-tNASP antibody or an anti-sNASP antibody may be essential, or both may be essential, and there is no limitation.

5. As described above, the composition for tumor diagnosis of the present invention is a composition comprising NASP (tNASP and sNASP) and / or TMF. That is, based on the knowledge that anti-NASP antibody or anti-TMF antibody is used as a tumor marker, NASP (NASP protein) or TMF (TMF protein) that can be used as an antigen as a means for detecting anti-NASP antibody and / or anti-TMF antibody Is used.

  Note that NASP and TMF in the composition of the present invention may be a recombinant protein prepared using a known gene recombination technique and a technique such as protein production and purification, or may be directly recovered and purified from a cell. (A protein derived from a living body) or a commercially available protein, not limited, and may be a part of NASP protein and TMF protein, in which case an antigenic determinant ( Peptide chains containing the epitope) are preferred. In this regard, the same definition explanation is applied to the present invention including NASP and TMF.

  Here, the composition for tumor diagnosis of the present invention may require either NASP or TMF, for example, for the purpose of detecting two or more tumors, etc. Not. When NASP is required, either tNASP or sNASP may be required, or both may be required and is not limited.

  Moreover, the tumor diagnostic composition of the present invention is preferably a tumor diagnostic agent. The present invention includes the use of NASP and TMF to produce a tumor diagnostic composition (preferably a tumor diagnostic agent).

(1) NASP and TMF
NASP and TMF used in the tumor diagnostic composition of the present invention can be prepared or obtained as follows, but are not limited thereto.

  As NASP and TMF, purified NASP and TMF can be used, but are not limited thereto. For example, an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the purified protein. And a protein having properties (activity, etc.) peculiar to NASP or TMF can also be used.

NASP amino acid sequence information is GenBank accession number (Accession No.).
It can be obtained from NM # 172164 (variant 1), NM # 002482 (variant 2) and NM # 152298 (variant 3).

(2) Mixing ratio of NASP and TMF In the diagnostic composition of the present invention, the mixing ratio of NASP and TMF is not particularly limited. For example, in the case of a tumor diagnostic agent or the like, the blending ratio of the NASP and TMF (the total ratio when there are a plurality of kinds) is preferably 1% by weight or more, more preferably 10% by weight. % By weight or more, more preferably 50% by weight or more, and even more preferably 80% by weight or more.

(3) Other components The diagnostic composition of the present invention may contain other components in addition to the NASP and TMF as long as the effects of the present invention are not significantly impaired, and is not limited. In the case of a tumor diagnostic agent or the like, for example, a reagent for detecting a primary antibody such as a chromogenic substrate can be included.

(4) Detection and quantification of anti-NASP antibody and / or anti-TMF antibody and evaluation of tumor status The diagnostic composition of the present invention (preferably tumor diagnostic agent) comprises anti-NASP antibody and serum in collected blood (serum). It is used to detect and quantify anti-TMF antibodies, and the possibility of various tumors and the degree of disease states can be judged (diagnosed) by comparison with the detected amount in healthy subjects. Moreover, the state of various tumors can also be determined by regularly monitoring the detected amount of the same patient.

(i) Detection of anti-NASP antibody and / or anti-TMF antibody Blood collected as a biological sample (test sample) has been subjected to treatment such as serum separation, and is generally 1 to 1 with a sample buffer such as PBS. Dilute 100 times.

  The biological sample after the treatment is brought into contact with the diagnostic composition of the present invention and used for the reaction with the NASP and TMF.

  Various reaction conditions such as the reaction temperature between the biological sample and the antibody and the reaction time are not limited, and may be set appropriately from a known condition range.

  The detection of anti-NASP antibody and / or anti-TMF antibody can be performed using a known immunoassay according to the conventional method. Immunoassays include labeled immunoassay and immunoturbidimetry (TIA), but the former is preferred.For example, in addition to enzyme immunoassay (EIA) such as ELISA, radioimmunoassay (RIA) And fluorescent immunoassay (FIA) are preferred. Further, as a labeled immunoassay method, a Western blot method (in combination with electrophoresis method) or the like is preferably used as a method utilizing a combination with other separation methods. Among these, ELISA and / or Western blotting are preferable, and ELISA is more preferable. In addition, one or two or more of the above antibodies may be used, and detection may be performed by combining two or more immunoassays, and excellent detection with higher sensitivity and reliability can be performed.

(ii) Quantification of anti-NASP antibody and / or anti-TMF antibody The quantification is not limited, but from a calibration curve based on the relationship between the amount of antibody (antibody concentration: concentration of anti-NASP antibody or anti-TMF antibody) and its detected value. Preferably, the anti-NASP antibody and / or anti-TMF antibody contained in the biological sample is quantified. The calibration curve is preferably prepared in advance based on a detection value (detection data) obtained using purified NASP or TMF and purified anti-NASP antibody or anti-TMF antibody. Specifically, information of detection values for each antibody concentration is obtained by the same method as the immunoassay (ELISA or the like) described above as the detection method, and prepared based on this. The amount of the anti-NASP antibody and / or anti-TMF antibody in the biological sample can be determined by comparing the calibration curve with the actual detection value (actual measurement value).

  Commercially available various kits can also be used for quantification or detection of anti-NASP antibody and / or anti-TMF antibody.

  The present invention also includes an anti-NASP antibody and / or anti-TMF antibody detection method characterized by reacting NASP and / or TMF with a biological sample as described above. The detection method may include a method for quantifying anti-NASP antibody and / or anti-TMF antibody, and is not limited.

(iii) Evaluation of tumor status In the present invention, the tumor status can be evaluated using the amount of anti-NASP antibody and / or anti-TMF antibody detected or quantitatively detected as described above as an index. “Evaluating the state of a tumor” means judging the presence / absence, progression, severity, treatment responsiveness, prognosis, etc. of a tumor (that is, (diagnosing a tumor). In addition to the NASP antibody amount and / or anti-TMF antibody amount, it may be performed in combination with subjective symptoms and various diagnostic imaging methods, etc. The anti-NASP antibody amount and the frequency of once or twice a year as in regular medical examinations. Although the tumor state may be evaluated by measuring the amount of the anti-TMF antibody, it is preferable to comprehensively evaluate by periodically monitoring the transition of the measured value at shorter intervals. For example, (a) the amount of anti-NASP antibody and / or the amount of anti-TMF antibody is preferably a constant value (in particular, the amount of anti-TMF antibody is preferably a constant value of 0 (may be a value below the detection limit)). ), It is judged (diagnosed) as a tumor that requires treatment, (b) already In the case of tumors being treated, if the amount of anti-NASP antibody and / or anti-TMF antibody is decreased, the treatment is diagnosed. (C) The amount of anti-NASP antibody and / or anti-TMF antibody is constant. It is possible to diagnose cases where the prognosis is poor for cases that exceed the threshold and change at a high level.

In addition, as described above, the present invention also includes a tumor diagnosis method characterized by using the detection result obtained by the detection method of the anti-NASP antibody amount and / or the anti-TMF antibody amount as an index.

6). Tumor diagnostic kit The tumor diagnostic kit of the present invention is a kit characterized by containing NASP and / or TMF, as described above, and can be used for carrying out the detection method and diagnostic method described above. .

  Here, in the tumor diagnostic kit of the present invention, either NASP or TMF may be essential, or both may be essential, and is not limited. When NASP is required, either tNASP or sNASP may be required, or both may be required and is not limited.

  The kit of the present invention that can easily and accurately measure an anti-NASP antibody and / or an anti-TMF antibody used as a novel tumor marker is expected to have great demand in the clinical practice of cancer treatment.

  In the kit of the present invention, the NASP and / or TMF is preferably provided in a dissolved state in consideration of stability (storage stability) and ease of use.

  The kit of the present invention can contain other components in addition to the NASP and / or TMF. Other components include, for example, an antigen-immobilized microplate, purified anti-NASP antibody and / or anti-TMF antibody (standard (STD)), antigen dilution, antibody dilution, HRP-labeled anti-mouse (or anti-mouse). Rabbit, anti-rat) IgG antibody, OPD (orthophenylenediamine) tablet, substrate solution (solution such as chromogenic substrate), reaction stop solution, concentrated washing solution, instruction manual (use manual) and the like.

  The kit of the present invention only needs to have at least the NASP and / or TMF as a component. Therefore, all of the components essential for detection of anti-NASP antibody and / or anti-TMF antibody or the like may or may not be provided together with the antibody, and there is no limitation.

  The kit of the present invention is required to be able to measure anti-NASP antibody and / or anti-TMF antibody with higher sensitivity, but in order to actually be useful in the clinical stage of cancer medical treatment, first, high measurement accuracy is required as performance. The reproducibility based on is further required.

  Measurement accuracy is an indicator that shows how much each measurement value varies when a single assay is performed in multiple test tubes or wells. Is expressed as a coefficient of variation (CV), that is, a ratio (%) of a standard deviation to an average value. In the kit of the present invention, this coefficient of variation (CV) is used as an index of reproducibility (simultaneous reproducibility), and the CV value is not limited, but is preferably 50% or less, more preferably 30% or less, for example. More preferably, it is 10% or less. In addition, regarding reproducibility (daily reproducibility) based on variations in measurement values for each measurement day, the CV value is preferably 50% or less, more preferably 30% or less, and even more preferably 10% or less.

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

1. "Cloning and identification of tumor antigen gene by SEREX method"
1-1. Method <Cell culture method>
VMRC-RCW, a renal cancer cell derived from an RCC patient, is a Minimum Essential Medium Alpha Medium with L-glutamine, ribonucleotides and deoxyribonucleotide (GIBCO), 10% FBS (Tissue Culture Biologicals), 1% Antibiotic Antimycotic ( GIBCO) was cultured under conditions of 37.0 ° C. and 5.0% CO ₂ .
<Human serum>
Serum used for screening for tumor antigens is a patient who participated in "Clinical research on autologous tumor cell inoculation with loss of GM-CSF gene transfer self-replication for stage IV renal cell carcinoma patients" The serum of the second case (hereinafter referred to as “GT2”) with the most clinical effect was used after obtaining written consent. Serum was collected on the 24th, 39th, 53th, 67th, and 81st days after the start of tumor vaccination, and sera mixed with equal amounts were used for screening. Serum was collected from various cancer-bearing patients and normal persons (healthy persons).
<Blocking and absorption of GT2 serum>
In 10 ml of LB medium with Carbenicillin, (1) BL21 (with pET / D / lacZ positive control plasmid or pBAD / D / lacZ positive control plasmid) is used for blocking and absorption of serum for Western blotting. 2) In the case of blocking and absorption of serum for SEREX, XL1-Blue MRF ′, an E. coli host, was inoculated and cultured overnight (250 rpm, 37.0 ° C.). The next day, 3 ml of E. coli that had been increased from the previous day was added to 300 ml of LB medium containing Carbenicillin and cultured until OD ₆₀₀ = 1.0 (250 rpm, 37.0 ° C), and 300 μl of 1M IPTG was added thereto and cultured for 4 hours (250 rpm, 37.0 ° C). After centrifuging this solution (3000 rpm, 15 min, 4 ° C.), the supernatant was discarded and suspended in 15 ml of TE. Thereafter, freezing at −80 ° C. and thawing at 37 ° C. were repeated 4 times in total, and ultrasonic crushing in ice water was repeated 6 times at 20 second intervals. This was centrifuged (10000 × g, 20 min, 4 ° C.), and the supernatant was transferred to a new tube to obtain an E. Coli lysate.

(1) For blocking and absorption of GT2 serum for Western blotting, mix 8.9 ml of 5% skim milk TBS-Tween (0.05% Tween) solution (0.1% NaN ₃ ) and 1 ml of E. Coli solution with 100 μl of serum. Incubation was performed overnight at 4 ° C., and the blocking treatment was terminated by centrifugation (3000 rpm, 10 min, 4 ° C.) the next day.

(2) For blocking and absorption of GT2 serum for SEREX, mix 4 ml of 5% skim milk TBS-Tween solution and 10 ml of E. Coli solution with 1 ml of serum, incubate overnight at 4 ° C, and centrifuge the next day ( 3000 rpm, 10 min, 4 ° C.) About 14 ml of the supernatant was transferred to a 100 ml bottle, and 86 ml of 5% skim milk was added thereto to make a total of 100 ml. Next, λZAP Express control DNA was subjected to absorption with a Gigapack (registered trademark) III Gold packaging extract phageized (negative phage). 1.5% NZY plate (171 mM NaCl, 16.2 mM MgSO ₄ · 7H ₂ O, 0.5% Yeast Extract, 1.0% NZ Amine (registered trademark), Type A, 1.5% Ager; Sumitomo Bakelite 150φ Petri dish for cell culture) per 2.0 × 10 ⁴ negative phage 2.0μl become pfu way ^{1.0 × 10 4 pfu / μl (} SM Buffer), LB solution of XL1-Blue MRF 'containing E. coli host (10 mM MgSO4, 0.2% maltose-containing OD ₍₆₀₀ = 0.5) Using 500 μl, collect 90 membranes in the same procedure as shown in the following SEREX section, collect them after treating with 100 ml of the primary antibody solution prepared earlier, and complete blocking and absorption. did.
<Extraction of RNA>
Each cell is cultured in a 150φ petri dish (Sumitomo Bakelite) for cell culture until just before the cells become saturated. After removing the culture solution, the cells are washed with 10 ml of PBS, and 18 ml of TRIzol (registered trademark) (Invitrogen) is added. Incubate for 5 minutes at room temperature, add 3.6 ml of CHCl ₃ , shake vigorously for 15 seconds, incubate for 3 minutes at room temperature, and centrifuge at 1750 × g, 4 ° C. for 15 minutes. The resulting aqueous layer was transferred to a new tube and 9 ml of iPrOH was added. Again shaken vigorously for 15 seconds, incubated at room temperature for 40 minutes, and centrifuged at 9600 × g, 4 ° C. for 20 minutes. The supernatant was discarded leaving the resulting pellet, washed with 18 ml of 80% EtOH, and centrifuged at 9600 × g and 4 ° C. for 5 minutes. The supernatant was discarded leaving the resulting pellet, dried for 10 minutes and then dissolved in sterile water. The RNA concentration was measured by measuring the absorbance of an RNA solution diluted 50 times using Multiskan Spectrum (Thermo Labsystems) at OD ₂₆₀ , and calculated by the formula of RNA (μg / μl) = (OD ₂₆₀ ) × 0.04 × D ( D is the dilution factor).
<Screening of tumor antigen gene expression by SEREX method>
The mRNA for preparing the phage library was extracted from cultured kidney cancer cells derived from VMRC-RCW and GT2 which are established kidney cancer cells. First, mRNA was extracted from the total RNA obtained in the above procedure according to the protocol attached to Fast Track (registered trademark) 2.0 kit (invitrogen), and ZAP Express (registered trademark) cDNA Synthesis Kit and ZAP Express (registered trademark) was used. (Trademark) cDNA was synthesized by cDNA Gigapack (registered trademark) III Gold Cloning Kit (STRATAGENE (registered trademark)), and an expression library was constructed using lambda ZAP Express, which is a lambda phage expression vector. 1 μl (SM Buffer) of a 10-fold diluted solution of this expression library was added to 200 μl of LB solution (10 mM MgSO ₄ , OD ₆₀₀ = 0.5 containing 0.2% maltose) in E. coli host XL1-Blue MRF 'for 15 minutes at 37.0 ° C. Incubation was carried out for infection, and the titer was measured by pouring the NZY plate with 2.5 ml of NZY top ager in solution at 42.0 ° C. The insert rate of the resulting plaques was examined by PCR directly on 40 plaques.

PCR is
Sense primer T3: 5'-AATTAACCCTCACTAAAGGG-3 '(SEQ ID NO: 1)
Antisense primer T7: 5'-GTAATACGACTCACTATAGGGC-3 '(SEQ ID NO: 2)
Using TOYOBO KODplus, the temperature was 94.0 ° C. for 2 min. (Denaturation 94.0 ° C. 30 sec. Annealing 56.0 ° C. 30 sec. Extension 68.0 ° C. 3 min.) × 35cycle 68.0 ° C. 5 min. Based on the results, adjust the mixing ratio of the phage solution and XL1-Blue MRF 'solution to 1.0 × 10 ⁴ pfu per NZY plate (150φ Petri dish, Sumitomo Bakelite), and incubate at 37.0 ° C for 15 minutes. Infected and poured into NZY plate with 6.5 ml of NZY top agar in solution at 42.0 ° C. Thereafter, it was dried at 42 ° C. for 30 minutes, and further incubated for 3.5 hours. After the plaque was confirmed, it was dried again for 30 minutes. Thereto, 20 mM IPTG aqueous solution was added and then dried Hybond ^™ -C Extra, Nitrocellulose membrane, 0.45 Micron, 137 mm (Amersham Biosciences) was attached to the plate surface and incubated at 37 ° C. for 3 hours. After 3 hours, peel off the membrane and wash twice with TBS-Tween (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween20) for 15 minutes with shaking to make 50 ml of 5% skim milk TBS-Tween solution. Soaked for 10 minutes and blocked overnight at 4 ° C. On the next day, the membrane was immersed in skim milk containing serum of the primary antibody that had been subjected to blocking treatment, and shaken for 4 hours. Serum was collected as much as possible and stored at 4 ° C. The membrane was washed twice with TBS-Tween and then shaken for 7 minutes three times. Next, 25 μl of secondary antibody (Alkaline phosphatase conjugated goat affinity purified antibody to human IgG Fc, ICN Pharmaceuticals, Inc.) was added to 50 ml of 5% skim milk TBS-Tween solution, and the membrane was soaked for 1 hour. After washing twice with TBS-Tween, shaking washing for 10 minutes was performed twice, and further, shaking washing for 7 minutes with TBS (without Tween) was performed twice, and Reaction Buffer (100 mM Tris-HCl (pH 9.5 ), 50 mM MgCl ₂ , 100 mM NaCl) for 20 minutes. The membrane subjected to the above operation was immersed in a coloring solution (Reaction Buffer 30 ml, NBT 13.5 mg, BCIP 5.25 mg; BIO-RAD) and appropriately transferred to distilled water while confirming the degree of dyeing. Thereafter, the membrane was washed twice with distilled water, soaked in TE for 10 minutes, and then the membrane was dried. Positive plaques were picked up based on the color development state of this membrane, each was dissolved in 200 μl of SM Buffer and vortexed, 5 μl of CHCl ₃ was added and stored at 4 ° C. Next, think that this phage recovered 90mm dish the same procedure as above using ^{(Falcon) Hybond TM -C Extra,} Nitrocellulose membrane, 0.45 Micron, performed in ^{82mm (Amersham Biosciences) (2 nd} screening), and positive plaques 4 plaques were picked up, the insert DNA part was amplified by PCR, and only those whose bands were confirmed at the same position by electrophoresis were identified as positive plaques and identified by DNA sequence analysis as SEREX antigen (Fig. 1) .

PCR is
Sense primer T3: 5'-AATTAACCCTCACTAAAGGG-3 '(SEQ ID NO: 1)
Antisense primer T7: 5'-GTAATACGACTCACTATAGGGC-3 '(SEQ ID NO: 2)
Was performed under the conditions of 94.0 ° C. 2 min, (denaturation 94.0 ° C. 30 sec. Annealing 56.0 ° C. 30 sec. Extension 68.0 ° C. 3 min.) × 34, 68.0 ° C. 5 min.
The sequence reaction is
Sense primer T3: 5'-AATTAACCCTCACTAAAGGG-3 '(SEQ ID NO: 1)
Antisense primer T7: 5'-GTAATACGACTCACTATAGGGC-3 '(SEQ ID NO: 2)
Using a Big Dye Terminator, the conditions were 96.0 ° C. for 2 min (denaturation 96.0 ° C. 30 sec. Annealing 50.0 ° C. 15 sec. Extension 60.0 ° C. 4 min.) × 25 cycles.
<DNA sequence analysis>
DNA amplified by PCR is purified by QIAquick PCR Purification Kit (QIAGEN), or DNA fragments with fluorescent material added are prepared by PCR using Applied Biosystems DNA Sequencing Kit using purified plasmids and phages as templates, and AutoSeq ^The PCR product was purified by ^TM G-50 (Amersham Biosciences). The purified product was subjected to DNA sequence analysis using ABI PRISM ^™ 377 DNA Sequencer. The gene corresponding to the obtained DNA sequence was searched by Nucleotide-nucleotide BLAST (blastn) on the NCBI website (http://www.ncbi.nih.gov/index.html).

1-2. Results As a result of immunoblotting using patient sera before and after treatment in the present inventor's previous report, strong reaction with the 250 kDa protein was observed in 4 cases. This signal appeared in the autologous serum after the treatment and could not be confirmed in the autologous serum before the treatment. Therefore, it was considered that the antibody against the protein was induced by the treatment. Based on this result, about 1 million pfu each using the VMRC-RCW mRNA and λ phage expression vector (Zap Express), which are the cultured renal cancer cells and established renal cancer cells of the second case who had a good clinical course An expression library was constructed, and the phage library was infected with E. coli as a host, and screening was performed by the SEREX method, which is one of Expression clonings using an antibody as a probe (FIG. 1). The serum of the patient of the second case was used as the primary antibody, anti-hIgG was used as the secondary antibody, and plaques that were more intensely colored than the surrounding were cloned as positive plaques. As a result, 12 SEREX antigens were isolated from the library derived from Case 2, 16 SEREX antigens were isolated from the library derived from VMRC-RCW, and a total of 28 positive plaques were obtained. It was found that C-terminal partial sequences of various known genes were cloned as antigens. Among them, the largest number was 9 cloned by tNASP. Below, there were 5 EKI1, 2 PHF3, vezatin, 2 E2b, and 1 other.

2. “Analysis of differential expression of SEREX antigen gene in normal / tumor cells in various tumor patients”
2-1. Method
Using BD Cancer Profiling Array II (BD Biosciences) as a cancer profiling array, the cDNA of each gene amplified by PCR is purified by 1% agarose gel electrophoresis and RECOCHIP (Takara) to form a probe, and for hybridization, Alkphos Direct Labeling Reagents (Amersham Bioscience) were used. PCR was performed using TOYOBO KODplus under conditions of 94.0 ° C. for 2 min. (Denaturation 94.0 ° C. 30 sec. Annealing 56.0 ° C. 30 sec. Extension 68.0 ° C. 3 min.) × 35 cycles 68.0 ° C. 5 min.

The primer of the probe cDNA used at this time was
NASP:
5'-CACCATGGCCATGGAGTCCACAGC-3 '(SEQ ID NO: 3)
5'- ACATGCAGTGCTTTCAACTGT-3 '(SEQ ID NO: 4)
TMF:
5'-CACCACAGTTGAATTTCTGAATGA-3 '(SEQ ID NO: 5)
5'-ACTGAGACTTTGTCTTAAAAGTTCA-3 '(SEQ ID NO: 6)
It is. (Template is a plasmid prepared for protein expression).

The membrane was previously immersed in milliQ water at room temperature, and prehybridization was performed at 55 ° C. for 30 minutes using a Hybridization Incubator (Robbins Scientific (registered trademark) MODEL1000; Robbins Scientific). The cDNA used was 100 ng, and the hybridization buffer was 12 ml. Hybridize overnight at 55.0 ° C with Hybridback (Cosmo Bio Inc.), then wash with primary wash buffer (2M urea, 0.1% SDS, 50 mM NaH ₂ PO ₄・ 2H ₂ O, 150 mM NaCl, 1 mM MgCl ₂ , 0.2% Blocking reagent) twice at 55 ° C for 10 minutes with shaking, and secondary washing buffer (50 mM Tris base, 100 mM NaCl, 2 mM MgCl ₂ , pH 10.0) twice at room temperature for 5 minutes with shaking The signal was detected by ECL plus Western Blotting Detection System (Amersham Biosciences). LAS-3000 (FUJIFILM) was used for signal detection. After detection of the signal, it was immersed in 800 ml of 0.5% SDS heated to 98.0 ° C. and left to reach room temperature for peeling treatment. In the array used this time, in 19 organs, that is, 10 each for breast, ovary, colon, stomach, lung, kidney, uterus, cervix, rectum, thyroid gland, testis, and skin, and 7 each for small intestine and pancreas A total of 154 data were examined for 5 people each in the bladder and vulva, 4 people in the prostate, and 3 people each in the trachea and liver. One block is for each organ, the left side in each block is from normal tissue, the right side is from cancer tissue, and the horizontal pair is from the same person.

2-2. Results The mRNA expression level of each SEREX antigen gene in normal and cancer tissues was examined using a Cancer profiling array. Each SEREX antigen cDNA was hybridized to the array, and the hybridization signal was detected by chemiluminescence. This signal was quantified to summarize which tissues had higher mRNA expression levels for each organ. The numerical value on the Y-axis represents the strength of cancer tissue corresponding to the strength of normal tissue as a logarithmic value. That is, a plot larger than 1 indicates that the expression level is high in cancer tissues, and a plot smaller than 1 indicates that the expression level is high in normal tissues. Each data is corrected by the intensity of ubiquitin. Those that showed a difference in the two-sided t-test of the two related groups are represented by red dots. As a result, it is the tNASP gene that attracts attention and tends to be highly expressed in cancer tissues in almost all organs except the thyroid gland, and significant in cancer tissues in five organs: ovary, uterus, cervix, testis, and small intestine The difference was recognized. However, other SEREX antigens did not tend to be biased to either one.

3. “Reactivity of antibodies in patient serum before and after vaccination with GM-CSF gene transfer”
3-1. <Construction of EREX antigen expression vector> (Fig. 2)
PET102 Directional TOPO (registered trademark) Expression Kits (Invitrogen) and pBAD102 Directional TOPO (registered trademark) Expression Kits (Invitrogen) were used to incorporate cDNAs of genes identified as SEREX antigens by SEREX into protein expression vectors. The cDNA to be incorporated into both vectors was synthesized in 20 μl by SuperScript ^TM First-Strand Synthesis System for RT-PCR (Invitrogen) using 3.6 μg of total RNA derived from VMRC-RCW obtained in the RNA extraction section. Then, 80 μl of sterilized water was added to make a total of 100 μl. Using this as a template, primers for each gene to be cloned were designed and amplified by PCR.

The primer used at this time was
NASP:
5'-CACCATGGCCATGGAGTCCACAGC-3 '(SEQ ID NO: 7)
5'- ACATGCAGTGCTTTCAACTGT-3 '(SEQ ID NO: 8)
TMF:
5'-CACCACAGTTGAATTTCTGAATGA-3 '(SEQ ID NO: 9)
5'-ACTGAGACTTTGTCTTAAAAGTTCA-3 '(SEQ ID NO: 10)
It is. A CACC sequence is added to the upstream 5 'side to provide directionality. The upper row is the upstream primer, and the lower row is the downstream primer.

PCR conditions were 94.0 ° C. for 2 min. (Denaturation 94.0 ° C. for 30 sec. Annealing 56.0 ° C. 30 sec. Extension 68.0 ° C. 3 min.) × 35 cycle 68.0 ° C. for 5 min. This was purified by QIAquick PCR Purification Kit (QIAGEN), 1% agarose gel electrophoresis and RECOCHIP (Takara). Incubated overnight at 37.0 ° C. to form single colonies. In order to check whether the desired plasmid is present in the resulting colony, colony PCR was performed using primers of the insert gene, and the plasmid that appeared to be positive was extracted by Rapid Plasmid Purification Systems (MARLIGEN). The DNA sequence was confirmed using ABI PRISM ^™ 377 DNA Sequencer as a template. The sequence reaction for sequence analysis of vector insert DNA is:
TrxFus Forward: 5'-TTCCTCGACGCTAACCTG-3 '(SEQ ID NO: 11)
Using a Big Dye Terminator, the conditions were 96.0 ° C. for 2 min (denaturation 96.0 ° C. 30 sec. Annealing 50.0 ° C. 15 sec. Extension 60.0 ° C. 4 min.) × 25 cycles.
<Purification of SEREX antigen protein>
The plasmid obtained by the above operation was transfected into E. coli host BL21 (DE3), and the entire amount was plated on an LB Ampicilline plate, and incubated at 37.0 ° C. overnight to form a single colony. Collect one colony with a yellow chip and incubate with 10 ml LB medium containing Ampicilline at 37.0 ° C, 250 rpm, overnight. Confluent BL21 (DE3) LB solution 1 ml and 30% glycerol LB solution 1 ml Was mixed well and stored at -80 ° C. Part of this BL21 (DE3) stored at -80 ° C is cultured in LB medium containing Ampicilline at OD ₆₀₀ = 1.0 at 37.0 ° C, 250 rpm. In the case of pET102 / D-TOPO, the final concentration is 1 mM. In the case of pBAD102 / D-TOPO, L-arabinose was added based on the results of the condition study (tNASP was a final concentration of 0.002% and sNASP was a final concentration of 0.02%). Thereafter, it was incubated at 37.0 ° C. and 250 rpm for 4 hours and exposed to ice water. When it was sufficiently cooled, it was transferred to a centrifuge tube, centrifuged at 3000 rpm, 4 ° C. for 10 minutes, and the supernatant was discarded. After cooling at −80 ° C. for about 20 minutes, the protein of interest was purified by thawing and using the MagneHis ™ Protein Purification Kit (Promega) protocol. At that time, 1/10 ml of Complete, Mini, EDTA-free (Roche) of Protease Inhibitor Cocktail Tablets was dissolved in Lysis Buffer and Elution Buffer used. The purified protein was measured for absorbance at OD ₇₅₀ using DC Protein Assay (BIO-RAD), and the concentration of the target protein was calculated from the calibration curve prepared based on the concentration and absorbance of the standard solution.
<SDS-PAGE>
Transfer the sample to a 1.5 ml Eppendorf tube and add an equal volume of 2x sample buffer (1.25 mM Tris-HCl (pH 6.8), 20% glycerol, 4% β-mercaptoethanol, 0.002 g / ml Bromophenol Blue, 0.04 g / ml SDS ) Was added, vortexed, boiled at 98 ° C. for 5 minutes, then exposed to ice water and then returned to room temperature. Next, remove the comb from the precast gel (PAG Mini “Daiichi” 10 (13w); Daiichi Kagaku) consisting of 4% concentrated gel and 10% acrylamide gel, and then the electrophoresis tank (Cassette electrophoresis tank “Daiichi”). “MODEL DPE-1020), and 400 ml of electrophoresis buffer (1 × Tris / glycine, 0.1% SDS) was poured. Flush the wells using a pipette, the sample and the marker (Precision Protein Standards ^TM or ^{Precision Plus Protein Standards TM; BIO-} RAD) using a capped the electrophoresis tank after applied POWER PAC3000 (BIO-RAD) 40 mA Electrophoresis was performed under conditions of 60 minutes.
<Western blotting>
The acrylamide gel after SDS-PAGE was immersed in a blotting buffer (1 × Tris / glycine, 20% MeOH) and shaken for about 20 minutes. In the meantime, the membrane (Immuno-Blot ^™ PVDF Membrane for Protein Blotting (0.2 μm); BIO-RAD) was permeabilized. First, the membrane was soaked in MeOH for 20 seconds with light shaking, immediately placed in a blotting buffer, and gently shaken for 5 minutes. Blotting filter paper (Extra Thick Blot Paper Mini blot size; BIO-RAD) was also placed in the blotting buffer and shaken gently. Next, prepare a semi-drive blotting device (TRANS-BLOT (registered trademark) SD SEMI-DRY TRANSFER CELL; BIO-RAD) and stack the filter paper, membrane, gel, filter paper, negative electrode in this order from the anode side. Energized in 60 minutes. After energization, shake with TBS-Tween (0.1% Tween) for 10 minutes, soak in 50 ml of 5% skimmed milk TBS-Tween (0.1% Tween) solution, shake the membrane at an appropriate location, and block overnight at 4 ° C. Went. On the next day, the blocking buffer was discarded, and lightly rinsed twice with TBS-Tween, and washed for 15 minutes, 5 minutes, and 5 minutes. Next, soak the membrane in the primary antibody solution for Western blotting prepared in the section on blocking and absorption of GT2 serum, shake at room temperature for 1 hour, rinse lightly twice with TBS-Tween, and then shake for 7 minutes. I went twice. Then, 2 μl of secondary antibody anti-hIgG (Peroxidase-Conjugated F (ab ') 2 Fragment of Rabbit Anti-Human IgG Specific for Gamma-Chains; DAKO) 10 ml of 5% skim milk TBS-Tween (0.05% Tween) The membrane was soaked in the solution and shaken at room temperature for 1 hour, rinsed lightly twice with TBS-Tween, and then shaken for 7 minutes 4 times.

  Then, arrange the membranes cut into strips on a flat wrap, absorb excess water with a Kimwipe, and use a detection solution that is a mixture of 5 ml of Solution A from ECL plus Western Blotting Detection System (Amersham Biosciences) and 125 μl of Solution B. The whole was incubated for 1 minute. After 1 minute, the excess detection solution was absorbed lightly with Kimwipe, wrapped in a wrap so as not to wrinkle, and then the band was detected with LAS3000 (FUJIFILM). After detection, place the membrane in antibody removal buffer (2% SDS, 100 mM β-Mercaptoethanol, 62.5 mM Tris-HCl (pH 6.8)) for reprobe treatment, incubate at 70 ° C for 30 minutes with shaking, and use TBS-Tween. Rinse twice twice, then shake twice for 7 minutes, soak in 5% skim milk TBS-Tween (0.1% Tween) solution and shake at room temperature for 1 hour, and add 0.5 μl of V5 antibody to 10 ml of 5% skim milk TBS. The membrane was immersed in a solution mixed with -Tween (0.05% Tween), shaken at room temperature for 1 hour, rinsed lightly twice with TBS-Tween, and then shaken for 7 minutes 4 times. Thereafter, the signal was again detected by ECL plus. LAS-3000 (FUJIFILM) was used for signal detection. In addition, when antibodies specific for each SEREX antigen were detected in the sera of various tumor patients, color development was detected with an NBT / BCIP solution using anti-hIgG (alkaline phosphatase conjugated; DAKO) as the secondary antibody.

3-2. Results Each SEREX antigen was incorporated into a pET or pBAD102 / D-TOPO protein expression vector in order to examine the reactivity of antibodies in patient serum to the SEREX antigen by Western blotting. This vector incorporates a His tag that enables purification of the expressed protein on the 3 'side of the cloning site and a V5 envelope that enables confirmation of band position and transcription amount. After electrophoresis on SDS-PAGE, it was transferred to a membrane and reacted with the serum before and after vaccination to quantify the antibody titer. As a result, an increase in antibody titer was confirmed after vaccination with tNASP and TMF. For each data, the antibody was removed by stripping after reaction with patient serum, reprobed with V5 antibody to confirm the position of the band, and corrected by the intensity of the reacted band.

4). “Confirmation of NASP and TMF gene expression using gene therapy patient samples”
4-1. Method Cultured normal kidney cells and cultured kidney cancer cells of the first case (GT1), second case (GT2), and third case (GT3) who participated in the clinical research of this GM-CSF immunogene therapy, the fourth case From the cultured kidney cancer cells of (GT4), the cultured normal kidney cells and cultured kidney cancer cells of GT3.1 entered, and VMRC-RCW, which is an established kidney cancer cell, were obtained by the procedure shown in the section of RNA extraction. Using 4.0 μg of each total RNA, cDNA was synthesized in a 20 μl system by SuperScript ^™ First-Strand Synthesis System for RT-PCR (Invitrogen), and 80 μl of sterilized water was added to make a total of 100 μl. Using this as a template, primers for tNASP, its splicing variant sNASP, and the TMF gene were designed and amplified by PCR. PCR was performed using TOYOBO KODplus under conditions of 94.0 ° C. for 2 min, (denaturation 94.0 ° C. for 30 sec. Annealing 56.0 ° C. 30 sec. Extension 68.0 ° C. 3 min.) × 30, 68.0 ° C. for 5 min.

The primer used at this time was
NASP:
5'-AGAACAGGTTTATGACGCCAT-3 '(SEQ ID NO: 12)
5'-CTTCATTAGCAGCTATCTGAA-3 '(SEQ ID NO: 13)
TMF:
5'-CACCACAGTTGAATTTCTGAATGA-3 '(SEQ ID NO: 14)
5'-ACTGAGACTTTGTCTTAAAAGTTCA-3 '(SEQ ID NO: 15)
The upper row is the upstream primer, and the lower row is the downstream primer.

4-2. result
Confirm whether tNASP, sNASP, and TMF are expressed in the Case 2 cultured renal cancer cells that were the library source, and also normal tissues and cancer tissues of three other stage IV renal cell carcinoma patients who were also vaccinated PT-PCR was performed in order to examine whether it was expressed in. For both tumor cells and normal tissue cells, in the search for gene expression in culture, all of tNASP, sNASP, and TMF were confirmed in both normal cells and cancer cells, and cancer-specific expression was observed in culture. There was no. (Figure 3)

5. “Confirmation of expression in each normal organ by cDNA panel of NASP and TMF genes”
5-1. Method
Using Human MTC ^TM Panel I, II (BD Bio sciences), each normal organ of tNASP, sNASP and TMF (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, In order to examine gene expression in the bladder, testis, uterus, small intestine, large intestine, and peripheral blood leukocytes), expression was confirmed by PCR. PCR was performed using TOYOBO KODplus under the conditions of 94.0 ° C. for 2 min, (denaturation 94.0 ° C. 30 sec. Annealing 56.0 ° C. 30 sec. Extension 68.0 ° C. 3 min.) × 34, 68.0 ° C. 5 min.

The primer used at this time was
NASP:
5'-AGAACAGGTTTATGACGCCAT-3 '(SEQ ID NO: 12)
5'-CTTCATTAGCAGCTATCTGAA-3 '(SEQ ID NO: 13)
TMF:
5'-CACCACAGTTGAATTTCTGAATGA-3 '(SEQ ID NO: 14)
5'-ACTGAGACTTTGTCTTAAAAGTTCA-3 '(SEQ ID NO: 15)
The upper row is the upstream primer, and the lower row is the downstream primer.

5-2. result
In SEREX antigen, tNASP, sNASP, and TMF, in which the amount of antibody was confirmed to be increased in the serum of patients after GM-CSF immune gene therapy compared to before treatment, each normal organ (heart, brain, placenta) RT-PCR was performed to confirm the expression status in lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, bladder, testis, uterus, small intestine, large intestine, and peripheral blood leukocytes). As a result, regarding all of tNASP, sNASP, and TMF, although there was a difference in the intensity of the expression level, the expression was confirmed in all tissues. (Figure 4)

6). "Reactivity of antibodies in the sera of various cancer patients"
The presence or absence of antibodies against SEREX antigen, NASP and TMF whose antibody levels had changed before and after GVAX inoculation were determined for various tumor patients (renal cell carcinoma, cervical cancer, colon cancer, endometrial cancer, melanoma, esophageal cancer, and lung cancer. ) And healthy individuals were examined by Western blotting, and the results were as shown in FIGS. Regarding tNASP (Fig. 5) that was most frequently cloned as a SEREX antigen, there was no particular difference in the presence or absence of the antibody between various tumor patients and healthy individuals. Interestingly, with regard to TMF (Fig. 7), antibodies were not detected in healthy individuals, showing a positive rate of 69% in renal cell carcinoma (antibody retention rate), 54% in cervical cancer, colon cancer 53%, endometrial cancer 47%, melanoma 39%, esophageal cancer 33%, lung cancer 13%.

7). "Discussion"
In order to identify cancer antigens in RCC, we have implemented the SEREX method, which is used as a technique for isolating cDNAs encoding cancer antigens recognized by IgG antibodies in the serum of cancer-bearing patients. We tried to identify cancer antigens with increased antibody titers before and after CSF gene vaccination. From the examination results by the SEREX method, 28 and 13 types of known gene clones were obtained.

  To compare the expression level of SEREX antigen mRNA in normal and cancerous tissues in various organs, we performed Cancer profiling array. As a result, there was a tendency for tNASP to be predominantly expressed in almost all organs. This NASP protein is present on chromosome 4 in a report in mice, and there are two variants of tNASP and sNASP, and sNASP has a structure in which the sequence of the central part of tNASP has been removed over about 1 kb by alternative splicing. Yes. Both have histone binding sites, leucine zippers, ATP / GTP binding sites, and nuclear localization signals. As for the expression tissue, tNASP is expressed only in testis, embryonic tissue and cancer cells, and sNASP is expressed in all dividing cells. In human NASP, there are variants 1 to 3 at the same locus on chromosome 1, variant 1 and 2 correspond to tNASP, and variant 3 corresponds to sNASP. As shown in FIG. 4, the expression in human normal tissues was confirmed in all tissues. In addition, RT-PCR was performed using cultured normal kidney cells and cultured kidney cancer cells, and expression was confirmed in both cells.

  When the reactivity of antibodies in patient sera before and after GM-CSF gene transfer vaccination against SEREX antigen was examined, an increase in antibody titer was confirmed after vaccination with tNASP and TMF. The calculated molecular weight of tNASP is 86.68 kDa and that of TMF is 120.23 kDa. In TMF, the amount of antibody increased about 10 times before and after vaccination. This showed that the increase in the amount of antibody against TMF was induced by GM-CSF gene-introduced vaccination and played a role in suppressing cancer growth. In addition, sNASP was a splicing variant of tNASP, and an interesting result was obtained that the reactivity of the antibody was high even though the sequence was all contained in tNASP. This is probably because an exon binding site different from tNASP is recognized.

  Furthermore, as a result of examining the presence or absence of antibodies in serum in various cancer patients and healthy individuals, TMF was not detected in healthy individuals and showed a positive rate of 69% in renal cell carcinoma, including 54% in cervical cancer, The results were 53% for colorectal cancer, 47% for endometrial cancer, 39% for melanoma, 33% for esophageal cancer, and 13% for lung cancer. TMF can be said to be particularly useful as a tumor marker because it is reactive only in cancer-bearing patients.

It is a figure which shows the outline of SEREX method. It is the schematic which shows the construction process of the expression vector of SEREX antigen. It is a photograph showing the results of confirming the expression of various mRNAs in normal cells and renal cancer cells. It is a photograph showing the results of confirming the expression of various mRNAs in various normal tissues. It is a graph which shows the antibody reaction positive rate in the serum of various tumor patients and a healthy person. It is a graph which shows the antibody reaction positive rate in the serum of various tumor patients and a healthy person. It is a graph which shows the antibody reaction positive rate in the serum of various tumor patients and a healthy person.

Sequence number 1: Primer sequence number 2: Primer sequence number 3: Primer sequence number 4: Primer sequence number 5: Primer sequence number 6: Primer sequence number 7: Primer sequence number 8: Primer sequence number 9: Primer sequence number 10: Primer Sequence number 11: Primer sequence number 12: Primer sequence number 13: Primer sequence number 14: Primer sequence number 15: Primer

Claims (9)

  A tumor marker comprising an anti-NASP antibody and / or an anti-TMF antibody.   2. The marker according to claim 1, wherein the tumor is at least one selected from the group consisting of renal cell cancer, colon cancer, melanoma, esophageal cancer, lung cancer, endometrial cancer, and cervical cancer.   A composition for tumor diagnosis, comprising NASP and / or TMF.   4. The composition according to claim 3, wherein the tumor is at least one selected from the group consisting of renal cell cancer, colon cancer, melanoma, esophageal cancer, lung cancer, endometrial cancer, and cervical cancer.   A tumor diagnostic kit comprising NASP and / or TMF.   6. The kit according to claim 5, wherein the tumor is at least one selected from the group consisting of renal cell cancer, colon cancer, melanoma, esophageal cancer, lung cancer, endometrial cancer, and cervical cancer.   A method for detecting anti-NASP antibody and / or anti-TMF antibody, comprising reacting NASP and / or TMF with a biological sample.   8. A method for diagnosing a tumor, wherein the detection result obtained by the method according to claim 7 is used as an index. 9. The method according to claim 8, wherein the tumor is at least one selected from the group consisting of renal cell cancer, colon cancer, melanoma, esophageal cancer, lung cancer, endometrial cancer, and cervical cancer.

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