Efficient Selection of WT1-Specific Donor Lymphocytes by Streptamer Technology.
Authors: Wang Xinchao , Schmitt Anita , Xu Xun , Chen Bao-An , Freund Mathias , Schmitt Michael 
Published: October 2019

Abstract
Abstract 3311
Poster Board III-199

BACKGROUND
After allogeneic hematopoietic stem cell transplantation, donor lymphocyte infusions (DLIs) have the potential to generate a desirable graft-versus-leukemia (GVL) effect, but bear the risk of eliciting a noxious graft-versus-host disease (GVHD). To minimize the risk of a GVHD and to improve the GVL effect, a positive selection of leukemia (antigen)-specific T cells would be highly desirable. In this study we focused on the leukemia-antigen Wilms Tumor gene 1 (WT1). In patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), a good correlation of WT1 expression and the number of leukemia blasts could be demonstrated. Several groups described immunogenic T cell epitopes derived from the WT1 protein.

MATERIAL AND METHODS
Here, we used the technology of streptamers available on a GMP level to detect the frequency of HLA-A2 restricted CD8+ T cells in the naïve peripheral blood (PB) from both healthy donors (HDs) and AML patients. Such WT1-specific CD8+ T cells were further characterized for the expression of CD27, CD28, CD45RA, CCR7 and CD107a. In the next step, WT1-specific cells were positive selected by magnetic cell separation (MACS™) columns after labeling with streptamers or tetramers and thereafter immunophenotyped. Moreover, mixed lymphocyte peptide cultures (MLPCs) were performed to enrich WT1-specific T cells derived from the PB of HDs. At last, WT1 specific T cells were evaluated in CFSE-based proliferation and cytotoxicity assays. Streptamer selected WT1-specific CD8+ T cells were compared with tetramer selected WT1-specific CD8+ T cells.

RESULTS
21 of 40 HDs showed naïve WT1 specific T cell frequencies of 0.5 to 2.0% of all CD8+ T cells. In two of ten AML patients, also 0.4 to 3.6% of WT1-specific T cells could be detected. Through streptamer based MACS™, a purity of more than 90% of all CD8+ T cells could be achieved for WT1-specific CD8+ T cells. These cells revealed to be CD8+WT1 _Streptamer+CD28+ CD45RA+CD69-CD107a+CCR7-CD137- effector T cells in flow cytometry. Proliferation assays proved that the streptamer technology does not alter the proliferative potential of the WT1-specific CD8+ T cells. In cytotoxicity assays, WT1-specific CD8+ T cells after streptamer selection were able to lyse HLA-A2+WT1+ cells at an effector/target ratio of 20:1. No significant difference between streptamer and tetramer selection could be found for the phenotype, as well as for the proliferative and cytotoxic potential of the cells.
After a maximum of three rounds of MLPC, only a frequency of 2-5% could be achieved, thus demonstrating the power of the streptamer technology.

CONCLUSION
In summary, the streptamer technology allows to select a highly purified fraction of WT1-specific effector T cells with proliferative and cytotoxic properties. In analogy to DLIs specific for viral antigens, production of leukemia specific DLIs is feasible on a GMP level. Further leukemia antigens are currently evaluated by our group.

Disclosures
No relevant conflicts of interest to declare.