Aurora kinase A-specific T-cell receptor gene transfer redirects T lymphocytes to display effective antileukemia reactivity

Abstract

IntroductionAurora kinase A (AURKA) is a member of the serine-threonine kinase family that regulates mitotic cell division from G 2 through to M phase of the cell cycle. 1 The AURKA gene maps to chromosome region 20q13.2. AURKA is expressed at low levels in normal cells, including dividing cells, and overexpression of AURKA has clear oncogenic potential. 2,3 Indeed, the AURKA gene is overexpressed in various types of cancer, 4 including leukemias. 5,6 Furthermore, correlations between the genetic dysregulation of AURKA and susceptibility to cancer, disease status, and prognosis have been described. 4 In particular, AURKA gene overexpression correlates with genetic instability and poor differentiation of cancer cells. 7,8 As AURKA expression is tightly regulated in normal tissues and overexpression correlates with malignant transformation, small molecular inhibitors have been developed that selectively target this protein in various tumors. A number of such molecules are currently in early phase clinical trials and preliminary data are encouraging. [9][10][11][12] The overexpression of AURKA in cancer cells, but not in normal tissues, makes it an attractive target for tumor immunotherapy. We have previously shown that testis is the only tissue that expresses detectable levels of AURKA, which suggests that this antigen behaves like cancer/testis antigens. 13 Based on these findings, we previously studied the immunotherapeutic potential of AURKA and identified an HLA-A*0201-restricted antigenic nonamer epitope derived from the kinase domain (residues 207-215). The AURKA 207-215 epitope (YLILEYAPL) was recognized by CD8 ϩ cytotoxic T lymphocytes (CTLs) generated in vitro. 6 Furthermore, leukemic cells endogenously expressing AURKA were killed by these CTLs, indicating that the cognate epitope is naturally processed and presented in the context of HLA-A*0201 at levels sufficient for immunotherapeutic applications. In addition, Kobayashi and colleagues have identified HLA-class II-restricted AURKA-derived pentadecamer epitopes to which they could generate CD4 ϩ helper T cells that expressed antitumor reactivity. 14 Immunotherapeutic interventions based on tumor antigenspecific T-cell receptor (TCR) gene transfer to redirect the specificity of other T cells has shown clinical success in patients with advanced melanoma. 15 However, this approach is complicated by several potential problems: (1) on-target adverse events directed against normal tissues, especially when affinity-enhanced TCRs are used 16 ; (2) issues related to chain mispairing between the introduced and endogenous TCR ␣/␤ genes; and (3) off-target adverse events because of inherent cross-reactivity of the introduced TCR. 17 Although various solutions have been explored to minimize TCR chain mispairing, all current approaches have intrinsic limitations. To this end, we have recently developed a unique vector system that simultaneously delivers siRNAs, which specifically down-regulate endogenous TCR expression, and a siRNA-resistant relevant TCR construct (...

Gene-Modified Human α/β-T Cells Expressing a Chimeric CD16-CD3ζ Receptor as Adoptively Transferable Effector Cells for Anticancer Monoclonal Antibody Therapy

“…þ Jurkat/MA/CD8a/ luc cells were isolated, expanded, and subjected to luciferase assay as described previously (28). One million CD16 þ Jurkat/ MA/CD8a/luc cells were coincubated with 1 Â 10 6 Raji cells or negative control K562 cells, with or without rituximab (1 mg/L), for 12 hours in advance at an effector:target ratio of 1:1.…”

“…After 4 days, CFSE dilution among cCD16z-T cells or NK cells was assessed by flow cytometry as described previously (28).…”

Antileukemia multifunctionality of CD4+ T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer

“…CD4 + T cells were cultured in GT-T503 medium (Takara Bio, Tsu, Japan) supplemented with 5% human serum, 0.2% human albumin, 50 U/ml recombinant human (rh) IL-2 (R&D Systems, Minneapolis, MN, USA) and 5 ng/ml interleukin-7 (IL-7; R&D Systems), and CD8 + T cells were cultured in the same medium additionally supplemented with 10 ng/ml IL-15 (PeproTech, Rocky Hill, NJ, USA) and 10 ng/ml IL-21 (Shenandoah Biotechnology, Warwick, PA, USA). CD4 + and CD8 + T cells were separately transfected with the same retroviral WT1-siTCR vector expressing the codon-optimized HLA-A*24:02-restricted and WT1 235-243 -specific TCR-α (Vα20/J33/Cα) and TCR-β (Vβ5.1/J2.1/Cβ2) genes derived from the cytotoxic CD8 + T-cell clone (TAK-1), 23,24 and siRNAs against endogenous TCR-α/β genes, in RetroNectin (Takara Bio)-coated plates as described previously. 19 Expression of WT1-specific TCR in WT1-siTCR/CD8 + T cells was assessed using an HLA-A*24:02/WT1 235-243 -tetramer.…”

Development of a novel redirected T-cell–based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia

“…36 Briefly, after complementary DNA was synthesized, qRT-PCR for hTERT mRNA (NM_198253) was performed by using the QuantiTect SYBR green PCR Kit (QIAGEN) and primers as follows: forward, 59-TTCTTGTTGGTGACACCTCACCTC-39; reverse, 59-CAGCCATACTCAGGGACACCTC-39 (Takara Bio). Human hypoxanthine phosphoribosyltransferase 1 (hHPRT1) mRNA (NM_000194) was prepared and used as an internal control.…”