Mitsuhiro Inoue

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.

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.

Identification of HLA-A2-restricted CTL epitopes of a novel tumour-associated antigen, KIF20A, overexpressed in pancreatic cancer

Background:Identification of tumour-associated antigens (TAAs) that induce cytotoxic T lymphocytes (CTLs) specific to cancer cells is critical for the development of anticancer immunotherapy. In this study, we aimed at identifying a novel TAA of pancreatic cancer for immunotherapy.Methods:On the basis of the genome-wide cDNA microarray analysis, we focused on KIF20A (also known as RAB6KIFL/MKlp2) as a candidate TAA in pancreatic cancer cells. The HLA-A2 (A*02:01)-restricted CTL epitopes of KIF20A were identified using HLA-A2 transgenic mice (Tgm) and the peptides were examined to check whether they could generate human CTLs exhibiting cytotoxic responses against KIF20A+, HLA-A2+ tumour cells in vitro.Results:KIF20A was overexpressed in pancreatic cancer and in some other malignancies, but not in their non-cancerous counterparts and many normal adult tissues. We found that KIF20A-2 (p12–20, LLSDDDVVV), KIF20A-8 (p809–817, CIAEQYHTV), and KIF20A-28 (p284–293, AQPDTAPLPV) peptides could induce HLA-A2-restricted CTLs in HLA-A2 Tgm without causing autoimmunity. Peptide-reactive human CTLs were generated from peripheral blood mononuclear cells of HLA-A2+ healthy donors by in vitro stimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2.Conclusion:KIF20A is a novel promising candidate for anticancer immunotherapeutic target for pancreatic cancers.

A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T-lymphocytes reactive to tumor cells

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)

Peptides derived from human insulin-like growth factor-II mRNA binding protein 3 can induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer cells

Insulin‐like growth factor‐II mRNA binding protein 3 (IMP‐3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP‐3 that can induce tumor‐reactive and human leukocyte antigen (HLA)‐A2 (A*02:01)‐restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP‐3‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their capacity to induce HLA‐A2‐restricted T cells in HLA‐A2.1 (HHD) transgenic mice (Tgm). We found that three IMP‐3 peptides primed HLA‐A2‐restricted CTL in the HLA‐A2.1 Tgm. Among them, human CTL lines reactive to IMP‐3 515NLSSAEVVV523 were reproducibly established from HLA‐A2‐positive healthy donors and lung cancer patients. On the other hand, IMP‐3 199RLLVPTQFV207 reproducibly induced IMP‐3‐specific and HLA‐A2‐restricted CTL from healthy donors, but did not sensitize CTL in the HLA‐A2.1 Tgm. Importantly, these two IMP‐3 peptide‐specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP‐3 and HLA‐A2. Cytotoxicity was significantly inhibited by anti‐HLA class I and anti‐HLA‐A2 monoclonal antibodies, but not by the anti‐HLA‐class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP‐3 protein was confirmed by specific killing of HLA‐A2‐positive IMP‐3‐transfectants but not the parental IMP‐negative cell line by peptide‐induced CTL. This suggests that these two IMP‐3‐derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy. (Cancer Sci 2011; 102: 71–80)

Identification of SPARC as a candidate target antigen for immunotherapy of various cancers

To establish efficient anticancer immunotherary, it is important to identify tumor‐associated antigens (TAAs) directing the immune system to attack cancer. A genome‐wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA‐A24 (A*2402)‐restricted and SPARC‐derived CTL epitopes. We previously identified H‐2Kd‐restricted and SPARC‐derived CTL epitope peptides in BALB/c mice, of which H‐2Kd‐binding peptide motif is comparable with that of HLA‐A24 binding peptides. By using these peptides, we tried to induce HLA‐A24 (A*2402)‐restricted and SPARC‐reactive human CTLs and demonstrated an antitumor immune response. The SPARC‐A24‐1143–151 (DYIGPCKYI) and SPARC‐A24‐4225–234 (MYIFPVHWQF) peptides‐reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA‐A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA‐A24 (A*2402). Furthermore, the adoptive transfer of the SPARC‐specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA‐A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.

An in vivo model of priming of antigen-specific human CTL by Mo-DC in NOD/Shi-scid IL2rγnull (NOG) mice

In vivo assay to evaluate anti-cancer immunotherapy at the pre-clinical phase is eagerly needed. We currently established xenotransplantation-based method to analyze in vivo priming of cancer-antigen-specific human cytotoxic T lymphocytes (CTLs). We transplanted human peripheral T cells and analyzed priming of CTLs in NOG mice. Half of the mice engrafted with bulk lymphocytes including CD4(+) T cells died before analysis probably due to xenoreactive graft versus host disease. All of the mice engrafted with purified CD8(+) T cells survived until the analysis, and successful engraftment was observed in 80% of recipient mice. Thus, transfer of purified CD8(+) T cells is sufficient and safer than that of bulk lymphocytes. To add antigenic stimulation to the CD8(+) T cells in vivo, injection of antigenic peptide-loaded and monocyte-derived autologous dendritic cells (DCs) was simultaneously done and repeated 7 days later. The DC-based vaccinization resulted in efficient priming of HLA class I-restricted and MART1, WT1 or CMV peptides-specific CTLs in the recipient mice. This system may be useful to evaluate the stimulation of antigen-specific human CTLs in vivo.

Abstract 4783: Identification of a novel TAA, SPARC, as a target for immunotherapy of various cancers

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC It is important to identify Tumor-associated antigens (TAAs) to direct the immune system to attack cancer in order to establish efficient anti-cancer-immunotherapy. We analyzed the gene expression profiles of gastric cancers by using the genome wide cDNA microarray consisting of probes for 20,340 genes, and found that SPARC (Secreted Protein Acidic and Rich in Cysteine) gene was overexpressed in diffuse-type gastric cancer tissues at higher levels in comparison to adjacent normal gastric tissues. The levels of expression of SPARC in pancreatic cancer and colorectal cancer tissues were also higher than those of normal counterparts. On the other hand, the expression of SPARC among the normal tissue specimens was detected only in limited tissue specimens at very low levels. Therefore, we evaluated the possibility of SPARC as a target antigen for anticancer immunotherapy. This study attempted to identify HLA-A24 (A*2402)-restricted and SPARC derived CTL epitopes. To identify the SPARC-derived and HLA-A24-restricted CTL epitopes, we selected a total of 4 different candidate 9- or 10- amino acid peptides that were expected to have a higher binding affinity to HLA-A24 (A*2402) by the HLA Peptide Binding Predictions in the BIMAS software program. The H-2 Kd-binding peptide motif is comparable to that of HLA-A24 binding peptide. Thus, we examined CTL response of BALB/c mice to these SPARC-derived epitopes. We found that two SPARC derived peptides elicited high magnitude of CTL response in BALB/c mice in an H-2Kd-restricted manner. Therefore, we attempted to generate SPARC-specific CTLs from the PBMCs of healthy donors and various cancer patients positive for HLA-A24 by stimulation of PBMCs with these two peptides. SPARC peptides-reactive CTLs were successfully induced from PBMCs by in vitro stimulation with these two peptides in HLA-A24 (A*2402) positive healthy donors and cancer patients. Moreover the SPARC-reactive CTLs established from PBMCs derived from cancer patients by stimulation with these two peptides, exhibited cytotoxicity to the cancer cells expressing both HLA-A24 and endogenous or transgene-derived SPARC. On the other hand, the CTLs did not kill the cells which were negative for either HLA-A24 or SPARC. Furthermore, the adoptive transfer of the SPARC-specific CTLs could inhibit the tumor growth in NOD/SCID mice bearing human cancer cells expressing both HLA-A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4783.