Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by reverse transcription polymerase chain reaction (RT-PCR) using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2'-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells, and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.
Background: Despite the development of novel drugs, including immune modulatory drugs (IMiDs) and proteasome inhibitors, multiple myeloma is still an uncurable disease. In particular, patients with high-risk chromosomal abnormalities, such as t (4;14) or del17, have poor prognosis. Recently, antibodies or CAR-T cells have been shown to be effective, suggesting that myeloma cells are sensitive to immune reactions. We have previously identified an HLA-A*24:02-restiricted epitope from a novel cancer-testis antigen CXorf48. In this study, we aim to characterize cytotoxic T lymphocyte (CTL) against this antigen and investigate the possibility of the CTLs for adoptive T-cell therapy in myeloma treatments. Methods: Expression of CXorf48 gene was examined by RT-PCR of myeloma cell lines. We then treated myeloma cells with low CXorf48 expressions by demethylating agents, 5-azacytidine (5-aza) or 5-aza-2’-deoxycytidine (DAC), and checked the expression level of this gene by quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors and their antigen recognition was examined by ELISpot assay and 51Cr cytotoxicity assay. We also analyzed T-cell receptor (TCR) repertoire using dextramer-sorted CTLs and transduced the TCR genes into Jurkat cells. Results: Expression of CXorf48 gene was found in most myeloma cell lines including those with t (4;14) or del17. In addition, the gene expression was significantly increased by DAC in CXorf48-low myeloma cells, which caused enhanced recognition by anti-CXorf48 CTLs. We also identified CXorf48-specidfic TCR genes of CTLs from two donors, and generated TCR gene-transduced T cells. Conclusions: CXorf48 might be a useful target for immunotherapy in combination with demethylating agents for high-risk myeloma. Aiming at CXorf48-specific adoptive therapy, we are analyzing CXorf48-specific TCR-transduced human T cells. Citation Format: Maiko Matsushita, Saku Saito, Kanae Mori, Shinya Yokoe, Daiju Ichikawa, Yutaka Hattori. A novel candidate for immunologic target in treatment of multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B73.
Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by RT-PCR using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2'-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.
Background and Objective: Development of drugs such as tyrosine kinase inhibitors (TKIs), immunomodulatory drugs (IMiDs), or proteasome inhibitors (PIs) have improved survival of hematologic malignancies including chronic myeloid leukemia (CML) and multiple myeloma (MM). However, some patients eventually relapse by discontinuation of the drugs or acquiring resistance to the drugs. Thus, it is crucial to develop novel therapy for cure of these malignancies. CML and MM are considered to be relatively immunogenic diseases among hematologic malignancies and immunotherapy might be useful. Since CML cells or MM cells have few somatic mutations which result in amino acid changes and give rise to neoantigens, checkpoint inhibitors alone may not be sufficient to evoke effective immunity against these cells. Instead, overexpressed antigen may be an attractive therapeutic target. We have identified CXorf48 as a highly expressed antigen in several hematologic malignancies including CML and MM. In this study, we investigated whether CXorf48 could serve as a therapeutic target for CML and MM. Methods and Results: High expression of CXorf48 gene in CML cells and MM cells was confirmed by quantitative PCR and also patients’ gene data obtained from a public repository. In contrast, BMMNCs cells or PBMNCs from healthy donor did not express CXorf48. We induced CXorf48-specific cytotoxic T lymphocytes (CTLs) by stimulating lymphocytes from healthy donors with HLA-A24:02-restricted CXorf48-derived epitope peptide. ELISpot assay, cytotoxic assay and CD107a mobilization assay showed that these CTLs preferentially recognized CML cells and MM cells. Moreover, we could detect CXorf48-specific CTL in peripheral blood of CML patients who remained in complete molecular remission after cessation of TKIs. Conclusion: Our data suggest that CXorf48 is a novel therapeutic target of CTLs in CML or MM. Immunotherapy against this antigen, such as vaccination therapy or adoptive T-cell therapy after conventional therapies might be an attractive therapeutic strategy for cure of CML and MM. Citation Format: Maiko Matsushita, Shinya Yokoe, Koji Ozawa, Saori Kanchi, Daiju Ichikawa, Eri Matsuki, Takehiko Mori, Shinichiro Okamoto, Yutaka Hattori. Evaluation of novel immune target in hematologic malignancies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A82.
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