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T Cells Genetically Engineered by HLA Class I-Restricted and WT1-Specific T-Cell Receptor Gene Transfer

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T Cells Genetically Engineered by HLA Class I-Restricted and WT1-Specific T-Cell Receptor Gene Transfer Leukemia (2015) 29, 2393–2401 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu ORIGINAL ARTICLE Antileukemia multifunctionality of CD4+ T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer H Fujiwara1, T Ochi1,2, F Ochi1,3, Y Miyazaki1, H Asai1, M Narita4, S Okamoto5, J Mineno5, K Kuzushima6, H Shiku7 and M Yasukawa1 To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4+ and CD8+ T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4+ and CD8+ T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms’ tumor 1 (WT1)-specific TCR-α/β genes and siRNAs for endogenous TCRs (WT1-siTCR/ CD4+ T cells and WT1-siTCR/CD8+ T cells), we examined the utility of this strategy. WT1-siTCR/CD4+ T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8+ T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4+ T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8+ T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4+ T cells and WT1-siTCR/CD8+ T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4+ T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8+ T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia. Leukemia (2015) 29, 2393–2401; doi:10.1038/leu.2015.155 INTRODUCTION tumor-specific helper T-cell function,13 employment of CD4+ Gene-modified T-cell-based antitumor adoptive immunotherapy T cells that have been gene modified to target well- now has a proven track record.1 For genetic retargeting of T cells characterized HLA class I-restricted tumor antigens would seem + toward predefined therapeutic antigens expressed by tumor cells, to be a more attractive option than ex vivo expansion of CD4 tumor antigen-specific T-cell receptor (TCR) gene or chimeric T cells by stimulation with HLA class II-restricted epitopes, as the antigen receptor (CAR) gene have been developed.2 Results from latter is too time-consuming and labor-intensive. However, the use preclinical and clinical studies using such ex vivo-expanded tumor- of gene-modified CD4+ T cells still remains at the preclinical – reactive T cells have revealed the prerequisite conditions for a stage,11,14 16 and to our knowledge, no study has yet investigated successful clinical outcome, and this has strengthened efforts to this approach for treatment of human leukemia. In the present improve the tumor-reactive effector functions mediated by gene- study, therefore, targeting Wilms’ tumor 1 (WT1), a well-studied modified CD8+ T cells.3 In particular, artificially mutated TCRs leukemia-associated antigen,17 we examined this concept with a conferring increased avidity on engineered CD8+ T cells4 and view to its possible clinical application. souped-up CAR constructs allowing prolonged in vivo survival5 For this purpose, we employed a high-performance HLA-A*24:02- have already been employed in clinical trials. However, such restricted and WT1235-243 nonamer epitope (CMTWNQMNL)-specific supraphysiologic functionality has resulted in lethal on- and off- TCR expression vector that simultaneously expresses siRNAs for target adverse events6,7 and cytokine release syndrome,8 both of constant regions of endogenous TCR-α/β genes in gene-modified which have emerged as issues of concern. Tcells(WT1-siTCR vector).18,19 In addition, we have recently Another reasonable approach for enhancing overall antitumor launched a clinical trial using WT1-siTCR gene-modified T cells for functionality mediated by therapeutically gene-modified CD8+ the treatment of patients with high-risk acute myelogenous T cells would be concomitant employment of tumor-reactive CD4+ leukemia and myelodysplastic syndrome (UMIN 0001159). T cells,9 as is the case with antiviral immunity.10 Because clinically In the present study, both CD4+ T and CD8+ T cells obtained effective HLA class II-restricted epitopes derived from tumor- from the same healthy individuals or patients with acute leukemia specific or tumor-associated antigens recognized by CD4+ T cells were gene modified using the same WT1-siTCR vector (to yield are limited in number,11 ex vivo-expanded tumor-reactive CD4+ WT1-siTCR/CD4+ T and WT1-siTCR/CD8+ T cells, respectively). We T cells have yet to be used clinically.12 To achieve effective then examined the influence of WT1-siTCR/CD4+ T cells on 1Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Toon, Ehime, Japan; 2Princess Margaret Cancer Center, Ontario Cancer Institute, Toronto, Ontario, Canada; 3Department of Pediatrics, Ehime University Graduate School of Medicine, Toon, Ehime, Japan; 4Laboratory of Hematology and Oncology, Graduate School of Health Science, Niigata University, Niigata, Japan; 5Center for Cell and Gene Therapy, Takara Bio Inc., Otsu, Shiga, Japan; 6Division of Immunology, Aichi Cancer Center, Nagoya, Aichi, Japan and 7Department of Cancer Vaccine and Immuno-Gene Therapy, Mie University Graduate School of Medicine, Tsu, Mie, Japan. Correspondence: H Fujiwara or M Yasukawa, Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Shitsukawa, Toon 791-0295, Ehime, Japan. E-mail: [email protected] or [email protected] Received 24 January 2015; revised 23 April 2015; accepted 1 May 2015; accepted article preview online 24 June 2015; advance online publication, 14 July2015 HLA class I-restricted WT1-specific CD4+ T cells H Fujiwara et al 2394 + + antileukemia functionality mediated by WT1-siTCR/CD8 T cells A24 or autologous CD14 monocytes untreated or pulsed with 0.1 μM WT1 both in vitro and in vivo. On the basis of our observations, we peptide (Greiner Bio-One, Frickenhausen, Germany) were co-cultivated assessed the clinical feasibility of using gene-modified HLA class with 2 × 105 WT1-siTCR/CD8+ T cells in the presence of WT1-siTCR/CD4+ I-restricted CD4+ T cells retargeted to WT1 in the context of T cells or NGM-CD4+ T cells in a 96-well plate along with anti-CD107a mAb gene-modified T-cell-based adoptive immunotherapy for human (BioLegend) for 3 h. Target-responsive interferon-γ (IFN-γ) production by + leukemia. WT1-siTCR/CD8 T cells was similarly assessed using an intracellular cytokine assay, as described previously.19 MATERIALS AND METHODS Cytokine secretion assay Cells Cytokines produced by WT1-siTCR/CD4+ T cells stimulated using the WT1 Approval for this study was obtained from the Institutional Review Board peptide-loaded C1R-A24 for 24 h were measured using a bead-based of Ehime University Hospital. Written informed consent was obtained from immunoassay kit (Becton Dickinson, San Jose, CA, USA). In some all patients and healthy volunteers in accordance with the Declaration of experiments, IFN-γ and IL-2 produced by WT1-siTCR/CD4+ T cells Helsinki. The HEK 293T (RIKEN BioResource Center, Tsukuba, Japan) was stimulated with leukemia cell lines or primary leukemia cells for 24 h used for lentiviral vector production. Epstein-Barr virus-immortalized were measured using an ELISA kit (Pierce, Grand Island, NY, USA). B-lymphoblastoid cell lines (B-LCLs), T2-A24,20 K562 (ATCC, Manassas, VA, Luminointensity was measured using IMMUNO-MINI (NJ-2300; Microtec, USA) and Jurkat (ATCC) were cultured as per normal. The HLA-A*24:02 Chiba, Japan). HLA class I or class II restriction was assessed similarly to the gene-transduced C1R (C1R-A24) and K562 (K562-A24) were maintained in a culture medium containing 0.5 mg/ml hygromycin B (Invitrogen, Carlsbad, cytotoxic T lymphocyte (CTL) assay. CA, USA) and 1.0 μg/ml puromycin (Sigma-Aldrich, St Louis, MO, USA), respectively. The HLA class II transactivator (CIITA) gene (EBO-76PL plasmid Carboxyfluorescein diacetate succinimidyl ester dilution assay encoding the CIITA-IRES-puromycin resistance gene,21 kindly provided by Dr To measure the target-responsive proliferation mediated by WT1-siTCR/ Viktor Steimle) was transduced into K562-A24 (K562-A24/CIITA) by CD8+ T cells, WT1-siTCR/CD8+ T cells labeled with a carboxyfluorescein electroporation and maintained using 0.5 mg/ml hygromycin B and 1 μg/ml diacetate succinimidyl ester (Molecular Probe Inc., Eugene, OR, USA) were puromycin. HLA class II genes expressed by K562-A24/CIITA were DR- + B1*03:01/04:04, DQ-B1*02:01/− and DP-B1*04:02/−. K562-A24/CIITA cells co-cultured for 3 days with WT1 peptide-loaded autologous CD14 lentivirally modified with the luciferase gene (K562-A24/CIITA/luc) were monocytes or primary leukemia cells in the presence of autologous + + 25,26 selected using blasticidin (Invitrogen). Peripheral blood mononuclear cells WT1-siTCR/CD4 T cells or NGM-CD4 T cells as described elsewhere. and bone marrow mononuclear cells from eight patients with acute + − myelogenous leukemia (six for HLA-A*24:02 and two for HLA-A*24:02 ), In vivo leukemia trafficking activity four patients with acute lymphoblastic leukemia (one for HLA-A*24:02+ − γ null 27 and three for HLA-A*24:02 ), one HLA-A*24:02+ patient with myeloid blast Six-week-old NOD/scid/ c (NOG) female mice were purchased crisis of chronic myelogenous leukemia (CML-BC) and healthy individuals from the Central Institute for Experimental Animals (Kawasaki, Japan) were isolated by density gradient centrifugation and stored in liquid and maintained in the institutional animal facility at Ehime University.
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