Citation: Kobayashi, E.; Kamihara, Y.; Arai, M.; Wada, A.; Kikuchi, S.; Hatano, R.; Iwao, N.; Susukida, T.; Ozawa, T.; Adachi, Y.; et al. Development of a Novel CD26-Targeted Chimeric Antigen Receptor T-Cell Therapy for CD26-Expressing T-Cell Malignancies. Cells 2023, 12, 2059. https://doi.org/10.3390/ cells12162059 Academic Editor: Subramaniam Malarkannan Received: 16 July 2023 Revised: 11 August 2023 Accepted: 11 August 2023 Published: 14 August 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Development of a Novel CD26-Targeted Chimeric Antigen Receptor T-Cell Therapy for CD26-Expressing T-Cell Malignancies Eiji Kobayashi 1,† , Yusuke Kamihara 2,† , Miho Arai 3 , Akinori Wada 2 , Shohei Kikuchi 2 , Ryo Hatano 4 , Noriaki Iwao 5 , Takeshi Susukida 6 , Tatsuhiko Ozawa 1 , Yuichi Adachi 3 , Hiroyuki Kishi 1 , Nam H. Dang 7 , Taketo Yamada 8 , Yoshihiro Hayakawa 6 , Chikao Morimoto 4 and Tsutomu Sato 2, * 1 Department of Immunology, Faculty of Medicine, Academic Assembly, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan; ekoba@med.u-toyama.ac.jp (E.K.); toz@med.u-toyama.ac.jp (T.O.); immkishi@med.u-toyama.ac.jp (H.K.) 2 Department of Hematology, Faculty of Medicine, Academic Assembly, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan; kamihara@med.u-toyama.ac.jp (Y.K.); akino@med.u-toyama.ac.jp (A.W.); skikuchi@med.u-toyama.ac.jp (S.K.) 3 Department of Pediatrics, Faculty of Medicine, Academic Assembly, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan; mkg8092@yahoo.co.jp (M.A.); ydachi@icloud.com (Y.A.) 4 Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan; rhatano@juntendo.ac.jp (R.H.); morimoto@ims.u-tokyo.ac.jp (C.M.) 5 Department of Hematology, Juntendo University Shizuoka Hospital, Izunokuni City, Shizuoka 410-2211, Japan; niwao@juntendo.ac.jp 6 Division of Host Defences, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan; susukida@inm.u-toyama.ac.jp (T.S.); haya@inm.u-toyama.ac.jp (Y.H.) 7 Division of Hematology/Oncology, University of Florida, Gainesville, FL 32610-0275, USA; namdang5@outlook.com 8 Department of Pathology, Saitama Medical University, 38 Morohongo, Moroyama, Saitama 3500495, Japan; taketo@keio.jp * Correspondence: tsutomus@med.u-toyama.ac.jp; Tel.: +81-76-434-7232 † These authors contributed equally to this work. Abstract: Chimeric-antigen-receptor (CAR) T-cell therapy for CD19-expressing B-cell malignancies is already widely adopted in clinical practice. On the other hand, the development of CAR-T-cell therapy for T-cell malignancies is in its nascent stage. One of the potential targets is CD26, to which we have developed and evaluated the efficacy and safety of the humanized monoclonal antibody YS110. We generated second (CD28) and third (CD28/4-1BB) generation CD26-targeted CAR-T-cells (CD26-2G/3G) using YS110 as the single-chain variable fragment. When co-cultured with CD26- overexpressing target cells, CD26-2G/3G strongly expressed the activation marker CD69 and secreted IFNgamma. In vitro studies targeting the T-cell leukemia cell line HSB2 showed that CD26-2G/3G exhibited significant anti-leukemia effects with the secretion of granzymeB, TNFα, and IL-8, with 3G being superior to 2G. CD26-2G/3G was also highly effective against T-cell lymphoma cells derived from patients. In an in vivo mouse model in which a T-cell lymphoma cell line, KARPAS299, was transplanted subcutaneously, CD26-3G inhibited tumor growth, whereas 2G had no effect. Furthermore, in a systemic dissemination model in which HSB2 was administered intravenously, CD26-3G inhibited tumor growth more potently than 2G, resulting in greater survival benefit. The third-generation CD26-targeted CAR-T-cell therapy may be a promising treatment modality for T-cell malignancies. Keywords: CD26; CAR-T; T-cell malignancies; third-generation; fratricide Cells 2023, 12, 2059. https://doi.org/10.3390/cells12162059 https://www.mdpi.com/journal/cells