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Background Aims: Hu8F4 is a T-cell receptor-like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor-expressing cells., Methods: We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor-expressing cells in vivo., Results: The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2 + PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice., Conclusions: Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo., Competing Interests: Declaration of competing interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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 MY8-specific 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., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood-derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci., Methods: We first determined the frequency of PR1-CTLs in HLA-A2(+) UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells., Results: PR1-CTLs are detected at an average frequency of 0.14% within the CD8(+) population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8(+) population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines., Conclusions: This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

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 into T 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 h8F4-CAR 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., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: Naturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1-5% of total nucleated cells in cord blood (CB) (<3 × 10⁶ cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed., Methods: Several methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHD mouse model., Results: With the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25⁺ cells. These were expanded to clinically relevant numbers by use of CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo-expanded Treg were CD4⁺25⁺ FOXP3⁺127(lo) and expressed a polyclonal T-cell receptor, Vβ repertoire. When compared with conventional T-lymphocytes (CD4⁺25⁻ cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro., Conclusions: In our NOD-SCID IL-2Rγ(null) xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo-expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: Tumor antigen-specific cytotoxic T lymphocytes (CTL) have been used in the treatment of human cancer, including leukemia. Several studies have established PR1 peptide, an HLA-A2.1-restricted peptide derived from proteinase 3 (P3), as a human leukemia-associated antigen. PR1-specific CTL elicited in vitro from healthy donors have been shown to lyse P3-expressing AML cells from patients. We investigated whether PR1-CTL can be adoptively transferred into NOD/SCID mice to eliminate human leukemia cells., Methods: PR1-CTL were generated in bulk culture from peripheral blood mononuclear cells (PBMC) stimulated with autologous dendritic cells. Human acute myeloid leukemia (AML) patient samples were injected and engrafted in murine bone marrow at 2 weeks post-transfer., Results: Following adoptive transfer, bone marrow aspirate from mice that received AML alone had 72-88% blasts in a hypercellular marrow, whereas mice that received AML plus PR1-CTL co-infusion had normal hematopoietic elements and only 3-18% blasts in a hypocellular marrow. The PR1-CTL persisted in the bone marrow and liver and maintained a CD45RA⁻CD28+ effector phenotype., Conclusions: We found that adoptive transfer of PR1-CTL generated in vitro is associated with reduced AML cells in NOD/SCID mice. PR1-CTL can migrate to the sites of disease and maintain their capacity to kill the AML cells. The surface phenotype of PR1-CTL was consistent with their trafficking pattern in both vascular and end-organ tissues.