Acute myeloid leukemia (AML) is a disease with poor prognosis. Fsm-like tyrosine kinase 3 (FLT3) is a promising target because of its overexpression in AML cells. Efforts have been put to develop new therapeutics targeting FLT3 by small molecule inhibitors and most recently with chimeric antigen receptor (CAR) modified T cells. We generated HLA-A2-restricted, FLT3-specific T cell receptors (TCR) to target FLT3-positive AML and hematopoietic stem cells (HSCs) in an HLA-A2-mismatched allogeneic-HSC transplantation. In our proposed set up, FLT3-specific TCRs would eliminate AML cells as well as HLA-A2-positive HSCs of the patient allowing engraftment of a healthy, HLA-A2-negative hematopoietic system. FLT3 is a self-antigen, therefore, T cells bearing high-affinity TCRs against epitopes derived from it are deleted in the thymus during T cell development. To circumvent the tolerance, we immunized a transgenic mouse model expressing a diverse human TCR repertoire and HLA-A2 molecule (ABabDII). The candidate epitopes for immunizations, FLT3839 and FLT3986, were selected among in silico predicted epitopes based on their binding affinity to HLA-A2 and homology to the mouse FLT3. We identified one TCR against FLT3839 (6546-IMS) and two TCRs against FLT3986 (6780-GLL and 6782-GLL). IFN- release was detected only from 6782-GLL T cells after overnight co-culture with a K562 cell line that was modified to express high levels of FLT3 and HLA-A2 proving FLT3986 epitope is naturally processed and presented. We tested the FLT3986-specific TCRs on three different cell lines that express FLT3 endogenously. We did not detect any CD137 upregulation by FACS or IFN- release by ELISA from neither of the FLT3986-specific TCRs against an AML cell line THP1. On the other hand, co-culture with SEM and MV-4;11 cell lines that express FLT3 endogenously and were modified to express HLA-A2 molecule, and with THP1 cells modified to overexpress FLT3 induced CD137 upregulation only on 6780-GLL T cells, but did not trigger any IFN- secretion suggesting higher FLT3 availability might be required for target cell recognition by the 6780-GLL TCR. This could be due to i) the sub-optimal avidities of the identified TCRs to the pMHC complex ii) low binding affinity of FLT3986 epitope to HLA-A2 molecule resulting in a poor presentation on the cell surface. In addition, recognition of MV-4;11 cells which carry the FLT3-ITD mutation suggested FLT3986 epitope is produced from both the wild type and mutated FLT3. During the in vitro safety testing, we discovered high, intracellular FLT3 expression in the Purkinje cells of the human cerebellum. We have stopped our attempt to identify high-affinity FLT3-specific TCRs due to potential cerebellar toxicity. We believe FLT3 could still be a safe, valuable target for therapies other than TCR-modified T cells.
