Leukaemia is the most common malignant disease of children in the United Kingdom,
Europe and the USA. Acute lymphoblastic leukaemia (ALL) is the most frequent
childhood leukaemia, with a 80-90% survival rate of 5 years. This project concers B
cell precursor ALL (BCP-ALL) the most common variant of ALL cases. Fusion of the
TEL (ETV6) and AML1 (RUNX1) giving rise to the TEL-AML1 chimaeric oncogene
are found in ~25% childhood BCP ALL cases. Expression of a novel TEL-AML1
oncoprotein by TEL-AML1+ BCP-ALL cells make this a possible target for HLArestricted
T cell immunity
The aim was to generate tumour cell lines that express HLA-A*02:01 in order to
generate a T cell response from HLA-A*02:01 restricted T cell receptors (TCRs). A
cDNA encoding HLA-A*02:01 was cloned into the mammalian vector (pcDNA3.1V5
His-A) in order to express this HLA-A*02:01 molecule in the TEL-AML+ HLAA0201-
BCP-ALL line REH and also control tumour cell lines including HT1080
(Fibrosarcoma) and LoVo (adenocarcinoma). Transfected cells were selected using the
co-expressed neomycin phosphotransferase gene in combination with the neomycin
analogue, G418. Initially, cell killing curves were generated to determine the sensitivity
of each cell line to G418.
In an alternative approach, a lentiviral vector expressing HLA-A*02:01 was also
generated. This lentiviral vector was generated by transient co-transfection of 293T
cells with the necessary packaging constructs and the generated vector used to
transduce target cell lines.
In order to confirm that the HLA-A*02:01 gene was expressed in a functional manner,
primary human T cells were transduced to express a recombinant HLA-A*02:01
restricted T cell receptor and expanded in culture to perform functional assays against
the HLA-A*02:01 expressing tumour cell lines.
Preliminary studies have used well characterised melanoma targets to functionally
validate the system (i.e. using the NY-ESO-1 target system). Future plans intend to
exploit the HLA-A*02:01 REH cells as a model to investigate CD8+ restricted T cell
responses against the TEL-AML1 antigen.