Background aims: Human myeloperoxidase has been shown to be overexpressed in many types of leukemia, such as chronic myeloid leukemia, acute myeloid leukemia and myelodysplastic syndrome. The authors identified two myeloperoxidase-derived HLA-A2-restricted peptides, MY4 and MY8, as novel leukemia-associated antigens. Methods: Ex vivo-elicited MY4-and MY8-specific cytotoxic T lymphocytes were generated, and tested for leukemia cell lysis in vitro and in NOD/SCID AML xenograft model. Results: These MY4-and MY8-specific cytotoxic T lymphocytes killed leukemic blasts while sparing healthy donor bone marrow cells. In addition, co-injection of MY4-and MY8-specific cytotoxic T lymphocytes into nonobese diabetic/severe combined immunodeficiency mice with acute myeloid leukemia drastically reduced tumor burden in vivo. The authors also found that MY4-and MY8-specific T cells could be detected in the peripheral blood mononuclear cells of allogeneic stem cell transplant recipients. Conclusions: These antigen-specific T cells were significantly increased in blood samples from patients compared with healthy donors, suggesting that both MY4 and MY8 are immunogenic and that MY4-and MY8specific cytotoxic T lymphocytes may play a role in reducing leukemia in vivo. Thus, the discovery of MY4 and MY8 as novel leukemia-associated antigens paves the way for targeting these antigens in immunotherapy against myeloid leukemia. (c) 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

The prognosis for patients with acute myeloid leukemia (AML) remains poor despite treatments. The need for new effective treatments in AML has led to the development of therapies that harness the immune system to attack the malignant cells. We have previously shown that neutrophil elastase (NE), proteinase 3 (P3) and cathepsin G (CG), serine proteases localized within azurophilic granules of myeloid cells, including myeloid leukemia, are immunotherapeutic targets for myeloid malignancies.

Background aims. The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4-chimeric antigen receptor (CAR) that was transduced intoT cells to mediate anti-leukemia activity. Methods. Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. Results. Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. Conclusions. Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.

The 5-year survival rate of acute myeloid leukemia (AML) patients is only about 25% because of the high relapse rate after standard chemotherapy; thus, effective new treatments such as immunotherapy are under study to improve survival. Preclinical and clinical studies have shown that adoptive cell therapy (ACT) utilizing gene-modified T cells expressing chimeric antigen receptors (CAR) has great potential for treatment of cancers, including leukemia. To design optimal CAR-modified T cells requires targeting a leukemia antigen that has been biologically well characterized. Studies have established the PR1 peptide, an HLA-A2.1-restricted peptide derived from proteinase 3 (P3) and neutrophil elastase (NE), as a human leukemia-associated antigen. We previously developed a T cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells and efficiently kills them in vitro and eliminates them in preclinical models. To study whether polyclonal T cells expressing CAR derived from the scFv of 8F4 would effectively target AML cells, we generated human T cells transduced to express PR1/HLA-A2 complex-specific CAR (8F4-CAR T) containing the scFv of 8F4 fused to the intracellular signaling endodomain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. We then tested 8F4-CAR T cells in in vitro and in vivo models. 8F4-CAR T cells demonstrated specificity and killed PR1-pulsed T2 cells but not control-peptide-pulsed T2 cells. 8F4-CAR T cells lysed HLA-A2 transduced U937 and HLA-A2+ primary AML from treatment-refractory patients but not HLA-A2-negative AML. To investigate the effects of 8F4-CAR T cells in vivo, we used a previously established PDX model of primary AML in sub-lethally irradiated NSG mice. The percentage of huCD45+ and HLA-A2+ cells in peripheral blood were used to confirm AML engraftment and to monitor leukemia growth. In addition, spleen, liver, lungs and bone marrow were harvested to assess leukemia burden when mice were sacrificed. A single dose of 8F4-CAR T cells resulted in expansion of the 8F4-CAR T cells in vivo and a marked and rapid loss of AML blasts in blood, bone marrow, spleen, liver, and lungs. We conclude that T cells expressing CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro and in vivo and this novel adoptive T cell approach merits further investigation.

PR1 is a HLA-A2–restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived APCs, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific CTLs (PR1-CTL) and the anti-PR1/HLA-A2 Ab 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an Ag become susceptible to therapies that target cross-presented Ags and suggest PR1 as a broadly expressed tumor Ag.