Abstract

Following allogeneic hematopoietic stem cell (HSC) transplantation, a graft versus leukemia (GVL) effect mediated by donor T cells contributes to the maintenance of long-term remission. GVL is likely mediated not only by alloreactive T cells, but also by donor lymphocytes recognizing tumor associated antigens overexpressed by leukemic cells. Selective expansion of such tumor-specific CTLs could augment GVL without increasing the risk of graft versus host disease (GVHD). PRAME is a cancer testis antigen (CTA) that is over-expressed by many hematological malignancies, including CML. We therefore generated PRAME-specific CTLs and evaluated their activity against primary CML tumor cells. CD8+ cells from 9 HLA-A*02 healthy donors and 5 CML patients were primed with autologous CD40L-activated B blasts loaded with HLA-A*02 restricted PRAME-peptides in the presence of low concentrations of IL-12, IL-7 and IL-15. Cells were then re-stimulated once a week using an artificial antigen presenting cell line (aAPC), consisting of K562 cell line that was genetically modified to stably express HLA-A*02, CD80 and CD40L and loaded with the same PRAME-peptides. In 8 out of 9 HLA A*02 healthy donors, we expanded (22±7 fold) T cells specific for the ALY-PRAME peptide. Similarly, PRAME-specific CTLs could be expanded from 3 out of 5 CML pts. When assessed by Elispot assay, these CTLs released IFNγ in response to ALY-peptide (511±260 SFC/105 cells) compared to 30±10 IFNγ SFC/105 against an irrelevant peptide. They also lysed autologous-PHA blasts loaded with ALY (84%±12% specific 51Cr release at 20:1 E:T ratio), but not with an irrelevant peptide (<10%). Both IFNγ release and cytotoxic activity were blocked by MHC class I antibodies, indicating that these effects were MHC-restricted. PRAME-specific CTLs also recognized primary CML cells since they produced IFNγ (320±31 SFC/105) in response to CD33+ CML blasts overexpressing PRAME (as assessed by Real Time PCR) isolated from HLA-A*02+ CML patients. Since routine expansion of PRAME-specific CTLs for clinical trials of adoptive T-cell transfer may remain problematic, we next cloned the αβT cell receptor (TCR) from a CTL clone with high affinity for the ALY-peptide (621 IFNγ SCF/105 cells at 0.2 nM peptide). The α- and β-chains were cloned in the SFG retroviral vector using an IRES sequence, and the transgenic αβTCR was expressed in primary T lymphocytes. T cells expressing the transgenic PRAME-specific αβTCR produced IFNγ on exposure to PRAME+ CML blasts (448± 69 IFNγ SFC/105 cells) and killed PHA blasts loaded with the ALY-peptide (42%±7% at 20:1 E:T ratio). In conclusion, our data show that PRAME is a promising target antigen on CML blasts and that adoptive transfer of CTLs or of T cells expressing a transgenic αβTCR targeting this antigen may be of value for patients with the disease.

Author notes

Disclosure:Research Funding: NIH and Leukemia & Lymphoma Society.