A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy.
Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene-transfer will be crucial for clinical applications of this technology.
Department of Hematology, Clinical Immunology and Infectious Disease, Ehime University Graduate School of Medicine, Ehime, Japan.
Nicholas Paul
NP
Casey
Department of Hematology, Clinical Immunology and Infectious Disease, Ehime University Graduate School of Medicine, Ehime, Japan.
Hiroshi
H
Fujiwara
Department of Hematology, Clinical Immunology and Infectious Disease, Ehime University Graduate School of Medicine, Ehime, Japan.
Kazushi
K
Tanimoto
CDM Centre, Takara Bio Inc., Shiga, Japan.
Sachiko
S
Okamoto
CDM Centre, Takara Bio Inc., Shiga, Japan.
Junichi
J
Mineno
Division of Immunology, Aichi Cancer Center, Aichi, Japan.
Kiyotaka
K
Kuzushima
Department of Cancer Vaccine and Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan.
Hiroshi
H
Shiku
Department of Hematology, Clinical Immunology and Infectious Disease, Ehime University Graduate School of Medicine, Ehime, Japan.
Masaki
M
Yasukawa
D064096:Aurora Kinase A / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D015536:Down-Regulation; D005822:Genetic Vectors / Q000494:pharmacology; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007938:Leukemia / Q000628:therapy*; D034741:RNA, Small Interfering / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics
D034741:RNA, Small Interfering; D011948:Receptors, Antigen, T-Cell; C576917:AURKA protein, human; D064096:Aurora Kinase A