Transcription factor Wilms' tumor gene 1 (WT1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in cancer progression. In clinical trials, WT1 is targeted using peptide-based or dendritic cell-based vaccines and T-cell receptor (TCR)-based therapies. Antitumor reactivities were reported, but T-cell reactivity is hampered by self-tolerance to WT1 and limited number of WT1 peptides, which were thus far selected based on HLA peptide binding algorithms.In this study, we have overcome both limitations by searching in the allogeneic T-cell repertoire of healthy donors for high-avidity WT1-specific T cells, specific for WT1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma samples.Using broad panels of malignant cells and healthy cell subsets, T-cell clones were selected that demonstrated potent and specific anti-WT1 T-cell reactivity against five of the eight newly identified WT1 peptides. Notably, T-cell clones for WT1 peptides previously used in clinical trials lacked reactivity against tumor cells, suggesting limited processing and presentation of these peptides. The TCR sequences of four T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against WT1-expressing solid tumor cell lines, primary acute myeloid leukemia (AML) blasts, and ovarian carcinoma patient samples.Our approach resulted in a set of naturally expressed WT1 peptides and four TCRs that are promising candidates for TCR gene transfer strategies in patients with WT1-expressing tumors, including AML and ovarian carcinoma.
<p>Supplemental Figure 1: RNA-sequence analysis allows discovery of antigens generated by exon skipping; Supplemental Figure 2: Sequence composition of the alternative HMSD transcript; Supplemental Figure 3: T-cells for LB-ITGB2-1 are detected after DLI; Supplemental Figure 4: Ex vivo activation of LB-ITGB2-1 specific T-cells; Table S1: predicted peptides for HMSD</p>
Allogeneic stem cell transplantation (alloSCT) provides a curative treatment option for hematological malignancies. After HLA-matched alloSCT, donor-derived T cells recognize minor histocompatibility antigens (MiHAs), which are polymorphic peptides presented by HLA on patient cells. MiHAs are absent on donor cells due to genetic differences between patient and donor. T cells targeting broadly expressed MiHAs induce graft-versus-leukemia (GvL) reactivity as well as graft-versus-host disease (GvHD), while T cells for MiHAs with restricted or preferential expression on hematopoietic or non-hematopoietic cells may skew responses toward GvL or GvHD, respectively. Besides tissue expression, overall strength of GvL and GvHD is also determined by T-cell frequencies against MiHAs. Here, we explored the use of DNA barcode-labeled peptide-MHC multimers to detect and monitor antigen-specific T cells for the recently expanded repertoire of HLA-I-restricted MiHAs. In 16 patients who experienced an immune response after donor lymphocyte infusion, variable T-cell frequencies up to 30.5% of CD8 + T cells were measured for 49 MiHAs. High T-cell frequencies above 1% were measured in 12 patients for 19 MiHAs, with the majority directed against mismatched MiHAs, typically 6–8 weeks after donor lymphocyte infusion and at the onset of GvHD. The 12 patients included 9 of 10 patients with severe GvHD, 2 of 3 patients with limited GvHD and 1 of 3 patients without GvHD. In conclusion, we demonstrated that barcoded peptide-MHC multimers reliably detect and allow monitoring for MiHA-specific T cells during treatment to investigate the kinetics of immune responses and their impact on development of GvL and GvHD after HLA-matched alloSCT.
Abstract Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8 + T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8 + T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo.
In Brief Alloreactive CD8+ T cells targeting minor histocompatibility antigens (MiHA) on malignant cells of the recipient play a pivotal role in graft-versus-tumor responses observed after allogeneic stem cell transplantation and donor lymphocyte infusion (DLI). However, these MiHA-specific CD8+ T-cell responses do not result in complete eradication of tumor cells in all patients. Furthermore, CD8+ memory T cells persisting after DLI do not always efficiently expand with recurrence of the disease. Adjuvant immunotherapy using dendritic cells (DC) loaded with hematopoietic-restricted MiHA may boost antitumor CD8+ T-cell immunity without inducing graft-versus-host disease. Here, we explored the use of mRNA-electroporated DC to stimulate MiHA-specific CD8+ T-cell responses. We demonstrate that electroporation of mature DC with P2X5 mRNA encoding for hematopoietic-restricted MiHA LRH-1 results in high expression of both mRNA and protein, and has no negative effect on the mature phenotype and migratory capacity of the DC. Furthermore, these DC can efficiently stimulate LRH-1–specific CD8+ effector T cells to proliferate and produce interferon-γ. In addition, LRH-1–specific CD8+ memory T cells that are present in patient-derived peripheral blood mononuclear cells at long periods post-DLI can be effectively activated by stimulation with P2X5 mRNA-electroporated DC to proliferate and degranulate upon target cell recognition. These results indicate that adjuvant immunotherapy using DC electroporated with mRNA encoding hematopoietic-restricted MiHA mismatched between patients and donors may enhance the graft versus tumor response induced by stem cell transplantation and DLI. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's web site, www.immunotherapy-journal.com
