Takuya Tsunoda

Identification of human leukocyte antigen-A24-restricted epitope peptides derived from gene products upregulated in lung and esophageal cancers as novel targets for immunotherapy

For the development of cancer vaccine therapies, we have searched for possible epitope peptides that can elicit cytotoxic T lymphocytes (CTL) to the TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K) and insulin‐like growth factor (IGF)‐II mRNA binding protein 3 (IMP‐3), which were previously identified to be transactivated in the majority of lung and esophageal cancers. We screened 31, 17 and 17 candidate human leukocyte antigen (HLA)‐A*2402‐binding peptides to parts of TTK, LY6K and IMP‐3, respectively. As a result, we successfully established strong CTL clones stimulated by TTK‐567 (SYRNEIAYL), LY6K‐177 (RYCNLEGPPI) and IMP‐3‐508 (KTVNELQNL) that have specific cytotoxic activities against the HLA‐A24‐positive target cells pulsed with the candidate peptides. Subsequent analysis of the CTL clones also revealed their cytotoxic activities against lung and esophageal tumor cells that endogenously express TTK, LY6K or IMP‐3. A cold target inhibition assay further confirmed that the CTL cell clones specifically recognized the MHC class I–peptide complex. Our results strongly imply that TTK, LY6K and IMP‐3 are novel tumor‐associated antigens recognized by CTL, and TTK‐567 (SYRNEIAYL), LY6K‐177 (RYCNLEGPPI) and IMP‐3‐508 (KTVNELQNL) are HLA‐A24‐restricted epitope peptides that can induce potent and specific immune responses against lung and esophageal cancer cells expressing TTK, LY6K and IMP‐3. (Cancer Sci 2007; 98: 1803–1808)

Vaccination with multiple peptides derived from novel cancer-testis antigens can induce specific T-cell responses and clinical responses in advanced esophageal cancer.

We previously identified three novel HLA‐A24‐restricted epitope peptides, which were derived from three cancer‐testis antigens, TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K), and insulin‐like growth factor (IGF)‐II mRNA binding protein 3 (IMP‐3), as targets for cancer vaccination against esophageal squamous cell carcinoma (ESCC). To examine the safety, immunogenicity, and antitumor effect of vaccine treatment using a combination of these three peptides, 10 HLA‐A2402‐positive advanced ESCC patients who failed to standard therapy were enrolled in a phase I clinical trial. Each of the three peptides (1 mg each) was intradermally administered with 1 mL of incomplete Freunds adjuvant to the neck in three separate regions weekly for 5 weeks. The cancer vaccination therapy was well tolerated without any treatment‐associated adverse events of grade 3 or 4. The TTK‐, LY6K‐, and/or IMP‐3‐specific T‐cell immune responses were observed by enzyme‐linked immunospot assay in peripheral blood lymphocytes obtained from nine of the 10 ESCC patients after their vaccination. The median survival time after the vaccination was 6.6 months. The vaccination could induce good clinical responses in 50% of the 10 patients. One patient experienced a complete response in hepatic metastasis lasting 7 months, one showed objective responses in all lung metastasis lesions, and three patients revealed a stable disease condition for at least 2.5 months. The cancer vaccine therapy using these three peptides demonstrated satisfactory safety and good immunogenicity as well as promising disease control rate, and therefore warrants further clinical studies. (Cancer Sci 2009)

Phase I clinical trial using peptide vaccine for human vascular endothelial growth factor receptor 2 in combination with gemcitabine for patients with advanced pancreatic cancer

Vascular endothelial growth factor receptor 2 (VEGFR2) is an essential factor in tumor angiogenesis and in the growth of pancreatic cancer. Immunotherapy using epitope peptide for VEGFR2 (VEGFR2‐169) that we identified previously is expected to improve the clinical outcome. Therefore, a phase I clinical trial combining of VEGFR2‐169 with gemcitabine was conducted for patients with advanced pancreatic cancer. Patients with metastatic and unresectable pancreatic cancer were eligible for the trial. Gemcitabine was administered at a dose of 1000 mg/m2 on days 1, 8, and 15 in a 28‐day cycle. The VEGFR2‐169 peptide was subcutaneously injected weekly in a dose‐escalation manner (doses of 0.5, 1, and 2 mg/body, six patients/one cohort). Safety and immunological parameters were assessed. No severe adverse effect of grade 4 or higher was observed. Of the 18 patients who completed at least one course of the treatment, 15 (83%) developed immunological reactions at the injection sites. Specific cytotoxic T lymphocytes (CTL) reacting to the VEGFR2‐169 peptide were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and the median overall survival time was 8.7 months. This combination therapy for pancreatic cancer patients was tolerable at all doses. Peptide‐specific CTL could be induced by the VEGFR2‐169 peptide vaccine at a high rate, even in combination with gemcitabine. From an immunological point of view, the optimal dose for further clinical trials might be 2 mg/body or higher. This trial was registered with ClinicalTrial.gov (no. NCT 00622622). (Cancer Sci 2009)

Rationale for Antiangiogenic Cancer Therapy with Vaccination Using Epitope Peptides Derived from Human Vascular Endothelial Growth Factor Receptor 2

Angiogenesis is a critical mechanism for tumor progression. Multiple studies have suggested that tumor growth can be suppressed if tumor angiogenesis can be inhibited using various types of antiangiogenic agents. Recent studies in mouse systems have shown that tumor angiogenesis can also be inhibited if cellular immune response could be induced against vascular endothelial growth factor receptor 2 (VEGFR2), which is one of the key factors in tumor angiogenesis. In this study, we examined the possibility of developing this novel immunotherapy in clinical setting. We first identified the epitope peptides of VEGFR2 and showed that stimulation using these peptides induces CTLs with potent cytotoxicity in the HLA class I-restricted fashion against not only peptide-pulsed target cells but also endothelial cells endogenously expressing VEGFR2. In A2/Kb transgenic mice that express alpha1 and alpha2 domains of human HLA-A*0201, vaccination using these epitope peptides in vivo was associated with significant suppression of the tumor growth and prolongation of the animal survival without fatal adverse effects. In antiangiogenesis assay, tumor-induced angiogenesis was significantly suppressed with the vaccination using these epitope peptides. Furthermore, CTLs specific to the epitope peptides were successfully induced in cancer patients, and the specificities of the CTLs were confirmed using functional and HLA-tetramer analysis. These results in vitro and in vivo strongly suggest that the epitope peptides derived from VEGFR2 could be used as the agents for antiangiogenic immunotherapy against cancer in clinical settings.

Identification of a Novel Tumor-Associated Antigen, Cadherin 3/P-Cadherin, as a Possible Target for Immunotherapy of Pancreatic, Gastric, and Colorectal Cancers

Purpose: To establish cancer immunotherapy, it is important to identify the tumor-associated antigens (TAA) that are strongly expressed in the tumor cells but not in the normal cells. In this study, to establish an effective anticancer immunotherapy, we tried to identify the useful TAA of pancreatic cancer. Experimental Design: Based on a previous genome-wide cDNA microarray analysis of pancreatic cancer, we focused on cadherin 3 (CDH3)/P-cadherin as a novel candidate TAA for anticancer immunotherapy. To identify the HLA-A2 (A*0201)–restricted CTL epitopes of CDH3, we used HLA-A2.1 (HHD) transgenic mice (Tgm). Furthermore, we examined the cytotoxicity against the tumor cells in vitro and in vivo of CTLs specific to CDH3 induced from HLA-A2–positive healthy donors and cancer patients. Results:CDH3 was overexpressed in the majority of pancreatic cancer and various other malignancies, including gastric and colorectal cancers, but not in their noncancerous counterparts or in many normal adult tissues. In the experiment using HLA-A2.1 Tgm, we found that the CDH3-4655-663 (FILPVLGAV) and CDH3-7757-765 (FIIENLKAA) peptides could induce HLA-A2–restricted CTLs in Tgm. In addition, peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A2–positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both CDH3 and HLA-A2. Furthermore, the adoptive transfer of the CDH3-specific CTLs could inhibit the tumor growth of human cancer cells engrafted into nonobese diabetic/severe combined immunodeficiency mice. Conclusions: These results suggest that CDH3 is a novel TAA useful for immunotherapy against a broad spectrum of cancers, including pancreatic cancer.

Multicenter, phase II clinical trial of cancer vaccination for advanced esophageal cancer with three peptides derived from novel cancer-testis antigens

BackgroundSince a phase I clinical trial using three HLA-A24-binding peptides from TTK protein kinase (TTK), lymphocyte antigen-6 complex locus K (LY6K), and insulin-like growth factor-II mRNA binding protein-3 (IMP3) had been shown to be promising for esophageal squamous cell carcinoma (ESCC), we further performed a multicenter, non-randomized phase II clinical trial.Patients and methodsSixty ESCC patients were enrolled to evaluate OS, PFS, immunological response employing ELISPOT and pentamer assays. Each of the three peptides was administered with IFA weekly. All patients received the vaccination without knowing an HLA-A type, and the HLA types were key-opened at the analysis point. Hence, the endpoints were set to evaluate differences between HLA-A*2402-positive (24(+)) and -negative (24(−)) groups.ResultsThe OS in the 24 (+) group (n = 35) tended to be better than that in the 24(−) group (n = 25) (MST 4.6 vs. 2.6 month, respectively, p = 0.121), although the difference was not statistically significant. However, the PFS in the 24(+) group was significantly better than that in the 24(−) group (p = 0.032). In the 24(+) group, ELISPOT assay indicated that the LY6K-, TTK-, and IMP3-specific CTL responses were observed after the vaccination in 63%, 45%, and 60% of the 24(+) group, respectively. The patients having LY6K-, TTK-, and IMP3-specific CTL responses revealed the better OS than those not having CTL induction, respectively. The patients showing the CTL induction for multiple peptides have better clinical responses.ConclusionsThe immune response induced by the vaccination could make the prognosis better for advanced ESCC patients.Trial registrationClinicalTrials.gov, number NCT00995358

Chemosensitivity testing of fresh human gastric cancer with highly purified tumour cells using the MTT assay.

A major problem associated with the chemosensitivity testing of fresh human tumour cells using the MTT assay is the contamination of nonmalignant cells in the tumour tissues. Highly purified fresh human gastric cancer cells could be obtained from 43 solid tumours and eight malignant ascites for the MTT assay. The success rate of the MTT assay was 87.9% (51 of the 58 cases), and the purity of tumour cells was greater than 90% after separation on Ficoll-Hypaque and Percoll discontinuous gradients in primary, or metastatic lesions, and also ascites. Cisplatin, mitomycin, and doxorubicin were more potent drugs than etoposide and 5-FU against gastric cancer cells. The chemosensitivity in differentiated cancer was equivalent to that in non-differentiated cancer. Twenty of the 51 patients with gastric cancer had evaluable lesions, and they received chemotherapy according to the results of the MTT assay using highly purified tumour cells. A clinical response was obtained in 12 of these 20 patients (response rate: 60.0%; five with complete response, seven with partial response).

Inhibition of Tumor Growth with Antiangiogenic Cancer Vaccine Using Epitope Peptides Derived from Human Vascular Endothelial Growth Factor Receptor 1

Purpose: Antiangiogenic therapy is now considered to be one of promising approaches to treat various types of cancer. In this study, we examined the possibility of developing antiangiogenic cancer vaccine targeting vascular endothelial growth factor receptor 1 (VEGFR1) overexpressed on endothelial cells of newly formed vessels in the tumor. Experimental Design: Epitope-candidate peptides were predicted from the amino acid sequence of VEGFR1 based on their theoretical binding affinities to the corresponding HLAs. The A2/Kb transgenic mice, which express the α1 and α2 domains of human HLA-A*0201, were immunized with the epitope candidates to examine their effects. We also examined whether these peptides could induce human CTLs specific to the target cells in vitro. Results: The CTL responses in A2/Kb transgenic mice were induced with vaccination using identified epitope peptides restricted to HLA-A*0201. Peptide-specific CTL clones were also induced in vitro with these identified epitope peptides from peripheral blood mononuclear cells donated by healthy volunteers with HLA-A*0201. We established CTL clones in vitro from human peripheral blood mononuclear cells with HLA-A*2402 as well. These CTL clones were shown to have potent cytotoxicities in a HLA class I–restricted manner not only against peptide-pulsed target cells but also against target cells endogenously expressing VEGFR1. Furthermore, immunization of A2/Kb transgenic mice with identified epitope peptides restricted to HLA-A*0201 was associated with significant suppression of tumor-induced angiogenesis and tumor growth without showing apparent adverse effects. Conclusions: These results strongly suggest that VEGFR1 is a promising target for antiangiogenic cancer vaccine and warrants further clinical development of this strategy.

HLA-A2-restricted CTL epitopes of a novel lung cancer-associated cancer testis antigen, cell division cycle associated 1, can induce tumor-reactive CTL

Toward the development of a novel cancer immunotherapy, we have previously identified several tumor‐associated antigens (TAAs) and the epitopes recognized by human histocompatibility leukocyte (HLA)‐A2/A24‐restricted cytotoxic T lymphocyte (CTL). In this study, we tried to identify a TAA of lung cancer (LC) and its HLA‐A2 restricted CTL epitopes to provide a target antigen useful for cancer immunotherapy of LC. We identified a novel cancer testis antigen, cell division cycle associated gene 1 (CDCA1), overexpressed in nonsmall cell LC using a cDNA microarray analysis. The expression levels of CDCA1 were also increased in the majority of small cell LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers. We used HLA‐A2.1 transgenic mice to identify the HLA‐A2 (A*0201)‐restricted CDCA1 epitopes recognized by mouse CTL, and we investigated whether these peptides could induce CDCA1‐reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA‐A2‐positive donors and a NSCLC patient. Consequently, we found that the CDCA165–73 (YMMPVNSEV) peptide and CDCA1351–359 (KLATAQFKI) peptide could induce peptide‐reactive CTLs in HLA‐A2.1 transgenic mice. In HLA‐A2+ donors, in vitro stimulation of PBMC with these peptides could induce peptide‐reactive CTLs which killed tumor cell lines endogenously expressing both HLA‐A2 and CDCA1. As a result, CDCA1 is a novel cancer‐testis antigen overexpressed in LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers, and CDCA1 may therefore be an ideal TAA useful for the diagnosis and immunotherapy of these cancers.

Detection of novel cancer‐testis antigen‐specific T‐cell responses in TIL, regional lymph nodes, and PBL in patients with esophageal squamous cell carcinoma

We recently identified three HLA‐A2402‐restricted epitope peptides derived from cancer‐testis antigens (CTA), TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K), and insulin‐like growth factor (IGF)‐II mRNA binding protein 3 (IMP‐3) for the development of immunotherapies against esophageal squamous cell carcinoma (ESCC). In order to evaluate their immunotherapeutic potential in ESCC patients, we estimated by ELISPOT assay the TTK‐, LY6K‐, or IMP‐3‐specific T‐cell immune responses in tumor‐infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL), and peripheral blood lymphocytes (PBL) expanded from 20HLA‐A2402 (+) ESCC patients, and correlated their immune activity with the expression levels of TTK, LY6K, and IMP‐3, and MHC class I in the tumors. Induction of TTK‐antigen specific T‐cell response in TIL to the peptide‐pulsed target cells was detected in 14 out of 20 (70%) cases, while LY6K or IMP‐3 specific T‐cell activity was observed in 11 of 20 (55%) or in eight of 20 (40%) cases, respectively. Furthermore, T‐cell activity in RLNL and PBL was detectable in the similar proportion of the 20 ESCC patients. Interestingly, CTA‐specific T‐cell immune response was found in 13 of 14 (93%) TIL obtained from ESCC tumors with strong MHC class I expression, while it could be observed only in two of six (33%) TIL from ESCC tumors with weak MHC class I expression. These results strongly suggest the pre‐existence of specific T‐cell responses to HLA‐A24‐restricted epitope peptides from TTK, LY6K, and IMP‐3 in ESCC patients. Monitoring antigen‐specific T‐cell responses, as well as the expression levels of MHC class I and epitope CTA in tumors, should be a selection index for application of cancer vaccine therapies to the patients who are likely to show good immune response. (Cancer Sci 2008; 99: 1448–1454)