Background and aim:
Adoptive T-cell therapies using synthetic chimeric antigen receptor expressing immune T-cells (CAR T-cells) have had unprecedented success in curing certain haematological malignancies. T-cell receptor (TCR) therapies are a new and evolving adoptive cell therapy, poised to become the next revolution in cancer treatment. TCR therapy can overcome some limitations of CAR therapy by enabling targeting of intracellular tumour antigens, extending the types of cancers that can be treated.
TCRs are highly specific for an antigen which is usually presented via a human leukocyte antigen (HLA) molecule. HLA alleles are highly polymorphic, and a range of HLA types are expressed in the Australian population, corresponding to our genetic diversity. Thus, generating a single TCR based therapy for an antigen will not be sufficient. This study aims to identify TCR sequences for the top HLA alleles and use genome editing tools to introduce them into normal healthy T-cells to generate novel therapeutic TCR T-cells.
Two important tumour antigens overexpressed in several malignancies including acute myeloid leukaemia, sarcoma, melanoma, glioma and ovarian cancers are Wilms tumour antigen1 (WT1) and Preferentially Expressed Antigen in MElanoma (PRAME), which are ideal candidates for TCR therapies.
Methods:
Peripheral blood mononuclear cells or cytotoxic T-cell (CTL) cultures were stimulated with antigenic WT1/PRAME peptides. Single reactive cells were captured using interferon-γ capture assays or tetramer staining, TCRαß sequences identified using 10X chromium single cell sequencing and modified with novel features to ensure precise pairing. TCRs were expressed in healthy donor T-cells using the piggyBac transposon system to generate transgenic TCR (tgTCR) T-cells. Functional responses were determined using intracellular cytokine flow and cytotoxicity assays, upon co-culture with HLA+ cells loaded with antigenic peptides. In silico analyses using x-scan mutagenesis are currently being undertaken to determine peptide binding affinity with mutations in the αßCDR3 regions, to facilitate future TCR affinity maturation optimisations. tgTCR specificity will be confirmed in vitro against primary tumours, cell lines, 3D spheroid models and in vivo in murine models.
Results and conclusion:
We have identified 4 functional HLA-A2 restricted TCRs against the WT-1 antigen. PRAME specific CTLs for HLA-A2/A1 have also been generated and potential epitopes identified using in silico analysis. We have developed an efficient and fast method to identify TCR sequences and generate tgTCR T-cells. We expect to develop a suite of tgTCR T-cells and establish off-the-shelf therapies to benefit a wide cross section of patients with different cancer types.