The present study attempted to identify a useful tumor-associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45-like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome-wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non-cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti-tumor effects of cytotoxic T-lymphocytes (CTL) specific to CDC45L-derived peptides induced from HLA-A24 (A*24:02)-positive donors. We identified three CDC45L-derived peptides that could reproducibly induce CDC45L-specific and HLA-A24-restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA-A24. In addition, we found that CDC45L 556KFLDALISL564 was eminent in that it induced not only HLA-A24 but also HLA-A2 (A*02:01)-restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L-specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L-derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy. (Cancer Sci 2011; 102: 697–705)
Lung cancer is the most common form of cancer and the leading cause of death from cancer-associated disease, accounting for 1.18 million of the 6.7 million cancer-related deaths worldwide.(1) Despite recent improvements in systemic therapy, the prognosis of patients with advanced-stage lung cancer remains very poor.(2) More effective treatment modalities are urgently required, and immunotherapy represents one promising approach for future lung cancer therapies.(3,4)
The success of therapeutic cancer vaccines may ultimately rely on the identification of immunogenic antigens that are overexpressed in tumors relative to normal tissues. Effective induction of cytotoxic T-lymphocytes (CTL) by tumor-associated antigen (TAA) has shown promising results.(5,6) Recently, the development of cDNA microarray technologies, coupled with genome information, has provided comprehensive profiles of the gene expression of malignant cells, which have been compared with those of normal cells.(7) Gene expression profiling with cDNA microarray technologies is an effective approach for the identification of new TAA useful for cancer diagnosis and immunotherapy.(8,9) By using this strategy, we focused on cell division cycle 45-like (CDC45L) as a novel TAA with possible utility in lung cancer immunotherapy.
The essential cellular protein CDC45L functions in both the initiation and elongation of DNA replication to ensure that chromosomal DNA is replicated only once per cell cycle.(10,11) The protein is absent in long-term quiescent, terminally differentiated and senescent human cells, although it is present throughout the cell cycle of proliferating cancer cells.(12) Thus, CDC45L expression is tightly associated with proliferating cell populations, and CDC45L is considered to be a promising candidate for a novel proliferation marker in cancer cell biology.(12,13) However, the usefulness of CDC45L as a target for cancer immunotherapy has not been previously investigated.
Materials and Methods
cDNA microarray analysis. Gene expression profiles were generated by cDNA microarray analysis, as described previously.(14,15) The raw data of microarray analysis is available upon request (Professor Y. Nakamura, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan). The tissue samples from lung cancers and adjacent non-cancerous normal lung tissues were obtained from surgical specimens, and all patients provided their written informed consent to participate in this study.
Patients, blood samples and tumor tissues. The research protocol for collecting and using PBMC from donors was approved by the Institutional Review Board of Kumamoto University. The blood samples or cancerous tissues and adjacent non-cancerous tissues were obtained from patients at Kumamoto University Hospital during routine diagnostic procedures after obtaining written informed consent. We also obtained blood samples from healthy donors after receiving their written informed consent. All samples were randomly coded to mask patient identities.
Peptides. Human CDC45L-derived peptides, carrying binding motifs for HLA-A24 (A*24:02)-encoded molecules, were selected using the BIMAS software program (BioInformatics and Molecular Analysis Section, Center for Information Technology, NIH, Bethesda, MD, USA), and 16 peptides (ten nonamers and six decamers, purity >95%) were synthesized (AnyGen, Gwangju, Korea) (Table S1). Peptides were dissolved in dimethylsulfoxide at the concentration of 20 μg/mL and stored at −80°C. Two HIV peptides, HLA-A24-presented RYLRDQQLL peptide (HIV-A24) and HLA-A2-presented SLYNTYATL peptide (HIV-A2), were used as negative controls.
Cell lines and HLA expression. The CDC45L and HLA-A24-positive human lung cancer cell lines EBC-1 and Lu99 were kindly provided by the Health Science Research Resources Bank (Tsukuba, Japan). The C1R-A2402 cells, an HLA-A24 transfectant of human B lymphoblastoid cell line C1R expressing a trace amount of intrinsic HLA class I molecule,(16) were a generous gift from Dr Masafumi Takiguchi (Kumamoto University, Kumamoto, Japan). The CDC45L-positive human pancreatic cancer cell line Panc1 (HLA-A2+, HLA-A24−) and the TAP-deficient and HLA-A2-positive cell line T2 were purchased from Riken Cell Bank (Tsukuba, Japan). The expression of HLA-A2 and HLA-A24 were examined by flow cytometry with an anti-HLA-A2 monoclonal antibody (mAb), BB7.2 (Abcam, Tokyo, Japan), and anti-HLA-A24 mAb (One Lambda Inc., Canoga Park, CA, USA), respectively, in order to select the HLA-A24- and HLA-A2-positive blood donors for the assays.
Reverse transcription-PCR and Northern blot analysis. The reverse transcription-PCR (RT-PCR) analysis of cell lines and normal or cancerous tissues was performed as described previously.(17) The CDC45L primer sequences were 5′-CTGGTGTTGCACAGGCTGTCATGG-3′ (sense) and 5′-CGCACACGGTTAGAAGAGGAG-3′ (antisense). A Northern blot analysis was performed as described previously using a CDC45L gene-specific cDNA probe (corresponding to 1245–1867 bp).(18)
Immunohistochemical staining. Immunohistochemical examination of human CDC45L was performed as described previously with some modification.(17) The detailed procedure of immunohistochemical examination is provided in Data S1.
Generation of CDC45L knockdown cells. To knock down the expression of CDC45L in lung cancer cells, CDC45L small interfering (si) RNAs (human Cdc45 siRNA, sc-35044: a pool of three target-specific 20–25 nt siRNAs; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were added at a final concentration of 150 nM to 40–60% confluent cells. Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, USA) was used to transfect the siRNA into cells, according to the manufacturer’s instructions. Green fluorescent protein siRNA were used as an irrelevant control. The treated cells were washed once with PBS, and adherent cells were collected at 72 h after transfection and used as target cells for the 51Cr-release assay. To investigate the ability of siRNA to suppress CDC45L expression, Western blot analysis was performed as described previously.(18) Cancer cells were washed once with PBS at 48 h after transfection, and adherent cells were collected and lysed to analyze the expression levels of CDC45L for comparison with those of negative control cells. β-Actin was used as the internal control. Rabbit polyclonal antibody reactive to CDC45L (sc-20685, Santa Cruz Biotechnology) was used as the primary antibody.
Immune response of CDC45L peptide-induced human CTL against cancer cell lines. The induction of CDC45L-reactive human CTL in vitro with CDC45L-derived peptides was performed as reported previously.(19,20) Six days after the last stimulation, the antigen-specific responses of the induced CTL were investigated. The frequency of cells producing interferon (IFN)-γ per 1 × 105 CTL upon stimulation with Lu99 cells (1 × 104 per well) or peptide-pulsed C1R-A2402 and T2 cells (1 × 104 per well) was analyzed by an ELISPOT assay (Human IFN-γ ELISPOT kit, BD Biosciences, San Jose, CA, USA) as previously described.(21) The CTL were co-cultured with cancer cells or peptide-pulsed C1R-A2402 and T2 cells as target cells (5 × 103 per well) at the indicated effector-to-target ratio, and a standard 51Cr-release assay was performed as described previously.(22,23) The blocking of HLA-class I by anti-human HLA class I mAb, W6/32 (IgG2a; Santa Cruz Biotechnology), or HLA-class II by anti-human HLA-DR mAb (IgG2a; BD Biosciences), was performed as described previously.(24,25)
CD107a mobilization assay. To identify degranulating CD8+ T-lymphocytes stimulated with epitope peptides, the CD107a exposed on the cell surface was analyzed by flow cytometry.(26,27) A CD107a mobilization assay was performed as described previously.(19) The CDC45L-derived peptide or control HIV peptide (1 μg/mL) was added as a stimulant.
Adoptive immunotherapy model. Experimental adoptive immunotherapy was performed as described previously.(28) Briefly, Lu99 cells (3 × 106 cells per mouse) positive for both endogenous CDC45L and HLA-A24 were subcutaneously inoculated into the right flanks of non-obese diabetic (NOD)/SCID mice. On day 7, when the tumor size reached approximately 25 mm2, the CDC45L-specific CTL lines or CTL lines induced by stimulation with HIV peptide were transferred. The CDC45L-specific CTL lines were established by stimulation of CD8+ T cells obtained from two healthy donors with a mixture of CDC45L-A24-2, -3 and -4 peptides. HIV-specific control CTL lines were established from CD8+ T cells from the same donors by the same culture procedure except that HLA-A24-presented HIV peptides instead of CDC45L peptides were added. The CTL lines derived from the two donors were suspended in PBS, and injected intravenously (4 × 106 cells/100 μL PBS per mouse). The intravenous injection of CTL was repeated on day 14. The tumor size was evaluated twice a week using calipers to measure two perpendicular diameters.
Identification of CDC45L gene overexpression in lung cancer based on cDNA microarray analyses. We used genome-wide cDNA microarray containing 27 648 genes to examine the gene expression profiles of 18 lung cancer tissues and their adjacent normal counterparts. The cDNA microarray analysis revealed markedly enhanced expression of the CDC45L gene in lung cancer tissues in all 12 of the small-cell lung cancer patients (average relative expression ratio, 163 000; range, 81 204–369 309) and four of the six non-small cell lung cancer patients (average relative expression ratio, 15 000; range, 0.08–40 131) (Table 1). Therefore, we selected CDC45L to be characterized as a novel TAA of lung cancer.
Table 1. Overexpression of CDC45L gene in lung cancer and various malignancies investigated by cDNA microarray analyses
Average of relative expression ratio
†The relative expression ratio (cancer/normal tissue) >5 was considered to be positive. ‡The tissue types of positive four cases were two adenocarcinoma and two squamous cell carcinomas. ‡The tissue types of negative two cases were one adenocarcinoma and one squamous cell carcinomas.
Small cell lung cancer
Urinary bladder cancer
Non-small cell lung cancer‡
Expression of CDC45L in normal organs, cancer cell lines and lung cancer tissues. The expression of CDC45L mRNA was analyzed by RT-PCR and Northern blot analysis. A semiquantitative RT-PCR analysis of CDC45L in normal tissues revealed that it was expressed only in testis (Fig. 1a). Likewise, Northern blot analysis revealed that CDC45L was not expressed in 22 normal adult organs except for testis (Fig. 1b). In contrast, strong expression of the CDC45L gene was detected in all nine lung cancer and various cancer cell lines by RT-PCR analysis (Fig. 1c). When we analyzed the expression of the CDC45L gene in lung cancer tissues by RT-PCR, we found strong expression of CDC45L mRNA in six of eight lung cancer patients (Fig. 1d, left). We also analyzed the expression of the CDC45L gene in lung cancer tissues and the adjacent normal tissues using RT-PCR. The expression of the CDC45L gene was detected in all seven lung cancer tissues, but little expression was detected in the adjacent normal tissues (Fig. 1d, right).
To investigate the expression of CDC45L at the protein level, we performed immunohistochemical analysis of lung cancer tissues and normal tissues. We studied 32 samples of lung cancer tissues, consisting of 12 adenocarcinomas (seven of the 12 were bronchioalveolar carcinomas), eight squamous cell carcinomas, six large cell carcinomas and six small cell carcinomas. All 32 samples exhibited strong nuclear staining of CDC45L and weak to strong cytoplasmic staining, but no staining or very weak staining was observed in normal adjacent lung tissues (Figs 2,S1). The CDC45L was expressed in testis, but no staining or weak staining was observed in other types of normal adult human tissues (Figs 2,S2). Collectively, the protein expression levels of CDC45L in human lung cancers were evidently much higher than those in normal adult tissues, with the exception of testis. These results are consistent with the results from RT-PCR and Northern blot analyses.
Screening for immunogenic CDC45L-derived peptides recognized by HLA-A24-restricted CTL. To identify immunogenic peptides derived from CDC45L, which can induce tumor-reactive and HLA-A24-restricted CTL, we selected 16 candidate peptides that were predicted to have high binding affinity to HLA-A24 according to HLA-peptide binding prediction software provided by the NIH BIMAS (Table S1). The CD8+ T cells sorted from the PBMC of two HLA-A24-positive healthy donors were stimulated with autologous monocyte-derived dendritic cells (DC) pulsed with a mixture of 4 of the 16 CDC45L peptides. The frequency of CD8+ T cells specific to the CDC45L-derived peptides in the resulting CTL lines was examined by an IFN-γ ELISPOT assay (Fig. 3a). Background controls were stimulated with C1R-A2402 cells pulsed with irrelevant HIV-A24 peptide. The generated CTL lines reproducibly produced a large amount of IFN-γ upon stimulation with C1R-A2402 cells pulsed with CDC45L-A24-2, -3, -4, -7 or -12 peptides. These results suggest that these five CDC45L-derived peptides are immunogenic.
To further analyze the CTL-stimulating capacity of these five immunogenic peptides, a CD107a mobilization assay was performed to evaluate the antigen-specific secretion of the cytolytic granule content by CTL.(26,27) A significantly higher proportion of CD8+ T cells was stained by anti-CD107a mAb when the CTL lines generated by stimulation with one of these five immunogenic peptides were re-stimulated with their cognate peptides, as compared to re-stimulation with an irrelevant HIV-A24 peptide (Fig. 3b).
Generation of CTL lines reactive to CDC45L-derived peptides in lung cancer patients. We generated CDC45L-specific CTL from the PBMC of lung cancer patients positive for HLA-A24 by stimulation with the CDC45L-A24-2, -3, -4, -7 or -12 peptides. These CTL lines produced a significantly large amount of IFN-γ in response to the cognate CDC45L-derived peptides in IFN-γ ELISPOT assays (Figs 4a,S3a). In addition, these CTL lines as well as the CTL lines induced from healthy donors exhibited cytotoxic activity against C1R-A2402 cells pulsed with the five CDC45L-derived peptides, but not against C1R-A2402 cells pulsed with irrelevant HIV-A24 peptide, in 51Cr-release assays (Figs 4b,c,S3b).
Natural processing of CDC45L CTL epitopes in cancer cells. We examined the ability of these CTL to kill human lung cancer cell lines that naturally expressed both CDC45L and HLA-A24. We used Lu99 and EBC-1 cells (CDC45L+, HLA-A24+), Lu99 and EBC-1 cells transfected with CDC45L-specific siRNA (CDC45L−, HLA-A24+), Lu99 and EBC-1 cells transfected with control GFP siRNA (CDC45L+, HLA-A24+) (Fig. 5a) and A549 cells (CDC45L+, HLA-A24−) as target cells. As shown in Figure 5b, the CTL lines generated from the healthy donor 4 (left) and lung cancer patient 18 (middle) by stimulation with CDC45L-A24-2 and -4 peptides, respectively, exhibited cytotoxicity against Lu99 cells and Lu99 cells transfected with control GFP siRNA, but not against Lu99 cells transfected with CDC45L-specific siRNA (left and middle) and A549 cells (left). Similarly, the CTL generated from lung cancer patient 17 by stimulation with CDC45L-A24-3 peptide exhibited cytotoxicity to EBC-1 and EBC-1 cells transfected with GFP siRNA, but not to EBC-1 cells transfected with CDC45L-specific siRNA and A549 cells (right). Among the five immunogenic CDC45L-derived peptides, three (CDC45L-A24-2, -3, and -4) elicited CDC45L-specific CTL that could effectively lyse lung cancer cells that naturally expressed both CDC45L and HLA-A24. These results suggest that these three CDC45L-derived peptides could be naturally processed and presented in the context of HLA-A24 molecules in cancer cells.
To confirm that the CTL specific to the three CDC45L-derived peptides recognize the target cells in an HLA-class I-restricted manner, we used mAb specific to HLA-class I (W6/32) to block the recognition by CTL. Production of IFN-γ (Fig. 5c) and cytotoxicity (Fig. 5d) were significantly inhibited by the blocking mAb against HLA-class I, but not by control anti-HLA-class II mAb. These results clearly indicate that these induced CTL recognize the target cells expressing endogenous CDC45L in an HLA-class I-restricted manner.
CDC45L-4 556KFLDALISL564 peptide can induce HLA-A2 (A*02:01)-restricted CTL. The CDC45L-A24-4 556KFLDALISL564 peptide was predicted to have a high binding affinity to not only HLA-A24 (A*24:02) but also HLA-A2 (A*02:01), according to HLA-peptide binding prediction software SYFPEITHI (Institute for Immunology, University of Tübingen, Tübingen, Germany, http://www.syfpeithi.de/). HLA-A24 is the most frequent HLA class I allele in the Japanese population, and HLA-A2 is one of the most common HLA alleles in various ethnic groups including Asian, African, Afro-American and Caucasian. Thus, we hypothesized that the CDC45L-A24-4 peptide is a common CTL epitope restricted by both HLA-A2 and HLA-A24. To determine whether the CDC45L-4 peptide can bind to HLA-A2 molecules, an HLA-A2-stabilizing assay was performed with T2 cells, as described previously.(23) The CDC45L-4 peptide bound to HLA-A2 molecules with a superior capacity to stabilize HLA-A2 compared to the HIV-A2 peptide, which was used as the positive control (data not shown). Thus, we confirmed the binding of the peptide to HLA-A2.
We next generated CDC45L-4-specific CTL from the PBMC of a healthy donor positive for HLA-A2 by stimulation with the CDC45L-4 peptide. The CTL lines generated from the HLA-A2-positive healthy donor produced IFN-γ specifically in response to re-stimulation with T2 cells pulsed with the peptide (Fig. 6a). In addition, the generated CTL lines exhibited cytotoxicity against T2 cells pulsed with the CDC45L-4 peptide, but not against T2 cells loaded with the irrelevant HIV-A2 peptide or C1R-A2402 cells loaded with the CDC45L-4 peptide (Fig. 6b). These results indicate that these CTL mediated peptide-specific cytotoxicity in an HLA-A2-restricted manner. Furthermore, the generated CTL lines could effectively lyse Panc1 cells that expressed endogenous CDC45L and HLA-A2 molecules but not HLA-A24, and the cytotoxicity was significantly inhibited by blocking mAb against HLA-class I (W6/32) but not by control anti-HLA-class II mAb, as determined by a 51Cr-release assay (Fig. 6c).
These results clearly indicate that CDC45L-4 peptide was naturally processed from CDC45L protein and presented not only in the context of HLA-A24 but also in the context of HLA-A2 to be recognized by CDC45L-4 peptide-induced CTL (Figs 4–6). Thus, this peptide will be applicable to immunotherapy for more than 75% of Japanese patients with lung cancer.
In vivo antitumor activity of CDC45L-reactive human CTL in NOD/SCID mice. To assess the therapeutic efficacy of CDC45L-reactive CTL inoculation into immunocompromised mice implanted with CDC45L-positive human lung cancer cells, we subcutaneously inoculated Lu99 cells into NOD/SCID mice. After 7 days, when the tumor diameters reached approximately 5 × 5 mm, mice were intravenously injected with human CTL generated by the stimulation of CD8+ T cells with autologous monocyte-derived DC (day 0) and autologous PHA-blasts (days 7, 14) pulsed with a mixture of CDC45L-A24-2, -3 and -4 peptides or an irrelevant HIV-A24 peptide. Before the inoculation of CTL into mice, we assessed the peptide-specific cytotoxic activity of CTL (Fig. S4a). The CTL lines generated from two HLA-A24-positive healthy donors produced IFN-γ specifically in response to re-stimulation with C1R-A2402 cells pulsed with the peptides, except for the CDC45L-A24-3 peptide in healthy donor 5. In addition, the mixture of CDC45L peptides elicited CTL that could effectively lyse Lu99 cells, and the cytotoxicity was significantly inhibited by blocking mAb specific to HLA-class I in a 51Cr-release assay (Fig. S4b).
The tumors in the mice inoculated with the CDC45L-stimulated CTL (n = 5; mean ± SD, 108 ± 65 mm2) were significantly smaller than those of mice inoculated with the control HIV peptide-induced CD8+ T cells (n = 5; mean ± SD, 271 ± 94 mm2) or with PBS alone (n = 5; mean ± SD, 297 ± 44 mm2) on day 42 after the inoculation of Lu99 cells (two-tailed Student’s t-test, *P < 0.05, **P < 0.01; Fig. 7). These results clearly indicate the efficacy of adoptive transfer therapy of CDC45L-specific human CTL against CDC45L-positive human tumors in NOD/SCID mice.
We identified a novel TAA, CDC45L, using cDNA microarray analysis of lung cancer tissues. In accordance with the data from the cDNA microarray analysis of CDC45L gene expression in lung cancer tissues, the expression of the CDC45L gene was detected in the majority of lung cancer tissues at both the mRNA and protein levels. In addition, the expression of the CDC45L gene at both the mRNA and protein levels was barely detectable in their normal counterparts and many adult organs, except for the testis, an immunologically privileged site. The frequent overexpression of CDC45L in lung cancers and the finding that novel immunogenic peptides can elicit an effective immune response against CDC45L-expressing tumor cells from CD8+ T cells of cancer-bearing patients suggest that CDC45L may be an antigen that is suitable for T cell-mediated immunotherapy.
We identified five CDC45L-derived peptides that were reproducibly able to induce peptide-specific CTL from CD8+ T cells isolated from both healthy donors and lung cancer patients. Three of these peptides, CDC45L-A24-2, -3 and -4, elicited CDC45L-specific CTL that could effectively lyse lung cancer cells endogenously expressing both CDC45L and HLA-A24. These results suggest that these three epitopes are naturally and efficiently processed from CDC45L protein in cancer cells, presented onto the cell surface in the context of HLA-A24 molecules and recognized by CTL. Thus, they might serve as a useful basis for CTL-mediated therapy.
We demonstrated that CDC45L-4 556KFLDALISL564 was an eminent and very rare epitope in that it induced not only HLA-A24 but also HLA-A2-restricted antigen-specific CTL. The allele HLA-A24 is one of the most common HLA-alleles in the Japanese population, with an estimated antigen frequency of 60%, and it is also present in 17% of Caucasians.(29) The allele HLA-A2 is one of the most common HLA alleles in various ethnic groups, and about 40% of the Japanese population is positive for HLA-A2.(30) Therefore, the CDC45L-4 peptide might be useful for the immunotherapy of many lung cancer patients worldwide.
Despite the self-protein nature of CDC45L, the CDC45L antigen is highly immunogenic. This result suggests that CDC45L may be a protein normally produced only in immunologically privileged sites or a protein normally not produced in an amount sufficient to be recognized by T cells.(31–33) Our results confirm these possibilities.
In conclusion, our results suggest that CDC45L antigen is highly immunogenic and a promising target for peptide-based immunotherapy of lung cancer. We expect that the combination of CDC45L-based immunotherapy with standard therapies, such as surgical operation, chemotherapy or radiation, will have enormous potential to improve the current outcomes of conventional cancer therapies.
We thank Drs Kentaro Yoshimoto, Kazunori Iwatani and Yasuomi Ohba (Department of Thoracic Surgery, Graduate School of Medical Sciences, Kumamoto University) for providing blood samples and tissues. We thank Drs Shinya Hirata, Tokunori Ikeda and Yusuke Matsunaga (Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University) for their technical advices. This work was funded by Grant-in-Aid (Nos 17015035, 18014023 and 22133005) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; a Research Grant for Health Sciences from the Ministry of Health, Labor and Welfare, Japan; and Funding from Onco Therapy Science Co. and from the Advanced Education Program for Integrated Clinical, Basic and Social Medicine, Graduate School of Medical Sciences, Kumamoto University (Program for Enhancing Systematic Education in Graduate Schools, MEXT, Japan).
This study was done in collaboration with Drs. Yataro Daigo and Yusuke Nakamura who are scientific advisors of the OncoTherapy Science, Inc.