Identification of HLA-A11 restricted T cell epitope of Wilms' tumor 1 (WT1) antigen and isolation of WT1-specific TCR

The WT1 protein encoded by the Wilms 'tumor 1(WT1) gene, is a tumor-associated antigen (TAA) and is overexpressed in a variety of hematopoietic malignant tumors and solid tumors. WT1 protein functions as a potent transcriptional regulator that regulates cell proliferation, cell death, and differentiation. WT1 protein is essential for the development of kidney, uterus, and gonad. Upregulated expression of WT1 on tumor cells is associated with aggressiveness and poor prognosis in multiple cancers, such as breast cancer, germ cell tumors, prostate cancer, and soft tissue sarcoma. In recent years, the development of tumor inununotherapy, especially adoptive T cell therapy (ACT), has substantially improved the prognosis of patients with metastatic cancer. Five CAR-T (chimeric antigen receptors-T) therapeutic agents have been commercially approved for clinical treatment of B-cell lymphoma or multiple myeloma. whereas CAR-T therapy is less effective for solid tumors. The T-cell receptor engineered T cell (TCR-T) therapy can target intracellular antigens of tumor cells presented by human leukocyte antigen (HLA) molecules through TCR, and has shown a tumorsuppressive effect on hematological and solid tumors in several clinical trials. A study of TCR-T cell therapy in acute myeloid leukemia (AML) patients found that adoptive infusion of TCR-T cells targeting the HLA-A2-restricted WT1 epitope into patients can effectively prevent AML relapse after bone marrow transplantation. HLA-A 11 is one of the most common HLA class I genotypes around the world, with a phenotypic frequency of about 20%-30% in the Chinese population. No HLA-A11-restricted T cell epitope has been reported for the WT1 antigen, which restricted the development of immune therapeutic agents for HLA-A11 patients. In this study, HLA-A11 restricted T cell epitopes of WT1 protein were in silico predicted and a CD8(+) T cell epitope WT1(414-423) (FSCRWPSCQK) was identified by immunizing HLA-A*11: 01 transgenic mice. A WT1(414-423) epitope-specific murine TCR, named 9E TCR, was obtained by WT1(414-423)/HLA-A11 tetramer staining and single cell sorting from mouse spleens. The cell based binding assays and affinity detection at the protein level showed that 9E TCR could specifically bind to WT1(414-423)/HLA-A11 with an affinity of 11.9 mu mol/L. In addition, 9E TCR-T cells were prepared by transducing primary T cells from HLA-A11(pos) healthy donors with 9E-TCR lentiviruses. 9E TCR-modified T cells targeting WT1(414-423) could specifically secrete IFN-gamma upon incubation with PANC-1 target cells presenting WT1(414-423) peptide, indicating that 9E TCR could mediate T cell activation in vitro upon recognition with WT1(414-423)/HLA-A11. The identification of WT1(414-423) epitope would provide a new target for tumor immunotherapy, and the isolation of 9E TCR that efficiently and selectively recognize WT1(414-423 )could benefit the future development of anti-tumor TCR-T cell therapeutic drugs. WT1-specific 9E TCR could represent an attractive therapeutic option for HLA-A11(pos) patients with high expression of WT1 protein in tumor tissues.