Discovering Optimal Targets for Adoptive T-Cell Immunotherapy of Leukemia.

Abstract 3016

Allogeneic hematopoietic cell transplantation is the most widely used form of adoptive T-cell cancer immunotherapy (ATCI) but its efficacy is limited by the fact that donor lymphocytes are neither selected nor primed (activated) prior to transfer. This can lead to tolerization of donor lymphocytes by tumor cells and causes graft-versus-host disease in 60% of recipients. These two caveats can be circumvented by the injection of primed antigen-specific CD8 T-cells targeted to tumor associated antigens (TAAs) or minor histocompatibility antigens (MiHAs). Several studies in humans have established the value of TAAs and MiHAs in therapies against solid tumors and leukemia, respectively. Importantly, the value of TAAs and MiHAs as targets for antigen-specific cancer immunotherapy has never been assessed against the same tumor.

Therefore, our main goal is to directly compare the therapeutic efficacy of T-cells targeted to TAAs vs. MiHAs. More specifically, we want to evaluate the in vivo anti-leukemic potential of MiHA- vs. TAA-primed CD8 T-cells and identify the mechanisms responsible for the differential anti-leukemic activity of antigen-specific T-cells.

We elected to work with 8 antigens known to be expressed on a mouse lymphoblastoma cell line (EL4 cells). Our panel includes 4 MiHAs of known sequence. We also selected 4 TAAs, whose sequences were elucidated by our group using a high-throughput mass spectrometry based approach (Fortier M.H. et al. 2008, J. Exp. Med). In vitro cytotoxicity assays revealed a functional CTL response against all of our antigens, thereby confirming their immunogenicity.

We first evaluated the anti-leukemic activity of antigen-specific CD8 T cells in vivo. Mice were immunized twice with peptide pulsed dendritic cells and challenged with 5 × 10⁵ EL4 cells 7 days after the last immunization. We observed an enhanced survival rate for mice immunized against MiHAs as opposed to mice immunized against TAAs. Indeed, while none of the mice immunized against TAAs survived the EL4 challenge, mice immunized against 3 out of the 4 MiHAs demonstrated either full survival (2 MiHAs) or partial survival with a delayed tumor onset.

To better understand the mechanisms responsible for the differential anti-leukemic activity of antigen-specific T-cells, we assessed the quality of binding of the 8 antigens to the MHC I molecule. More specifically, we measured the half-life of MHC I/peptide complexes at the cell surface and performed a peptide binding competition assay to evaluate the affinity of our 8 antigens for MHC I molecules. Notably, MiHAs and TAAs showed comparable half-life of MHC I/peptide complexes at the cell surface and binding affinities. We conclude that differential MHC I/peptide interactions are not responsible for the differential anti-leukemic activity of MiHA- vs. TAA-primed CD8 T-cells.

We then investigated the frequency of antigen-specific CD8 T-cells in immunized mice using MHC class I tetramers. Our preliminary results show that CD8 T-cells specific for 4 of our antigens (3 TAAs and 1 MiHA) are undetectable by flow cytometry. Interestingly, we found a strong correlation between the lack of tetramer staining and lack of survival of EL4 bearing mice. This suggests that CD8 T-cells targeting TAAs bind weakly to their respective MHC I/peptide complexes, which could indicate a weaker immunogenicity compared to MiHAs. It will be interesting to investigate whether and how other mechanisms of immunogenicity, such as TCR avidity and T-cell frequency, can influence the outcome of antigen-specific leukemia immunotherapy.

Our studies will provide the first direct comparison of the anti-leukemic potential of MiHA- vs. TAA-primed CD8 T-cells. We believe that these crucial informations will serve as guide for selecting the best antigens for antigen-specific ATCI in future clinical trials.