RESUMO
Relapse is the leading cause of death after allogeneic hematopoietic stem cell transplantation (HCT) for leukemia. T cells engineered by gene transfer to express T cell receptors (TCR; TCR-T) specific for hematopoietic-restricted minor histocompatibility (H) antigens may provide a potent selective anti-leukemic effect post-HCT. We conducted a phase I clinical trial employing a novel TCR-T product targeting the minor H antigen HA-1 to treat or consolidate treatment of persistent or recurrent leukemia and myeloid neoplasms. The primary objective was to evaluate the feasibility and safety of administration of HA-1 TCR-T post-HCT. CD8+ and CD4+ T cells expressing the HA-1 TCR and a CD8-co-receptor were successfully manufactured from HA-1 disparate HCT donors. One or more infusions of HA-1 TCR-T following lymphodepleting chemotherapy were administered to nine HCT recipients who had developed disease recurrence post-HCT. TCR-T cells expanded and persisted in vivo after adoptive transfer. No dose-limiting toxicities occurred. Although the study was not designed to assess efficacy, four patients achieved or maintained complete remissions following lymphodepletion and HA-1 TCR-T, with one ongoing at >2 years. Single-cell RNA sequencing of relapsing/progressive leukemia after TCR-T therapy identified upregulated molecules associated with T cell dysfunction or cancer cell survival. HA-1 TCR-T therapy appears feasible and safe and shows preliminary signals of efficacy. This clinical trial is registered at clinicaltrials.gov as NCT03326921.
RESUMO
Leukemia relapse remains the major cause of allogeneic hematopoietic stem cell transplantation (HCT) failure, and the prognosis for patients with post-HCT relapse is poor. There is compelling evidence that potent selective antileukemic effects can be delivered by donor T cells specific for particular minor histocompatibility (H) antigens. Thus, T-cell receptors (TCRs) isolated from minor H antigen-specific T cells represent an untapped resource for developing targeted T-cell immunotherapy to manage post-HCT leukemic relapse. Recognizing that several elements may be crucial to the efficacy and safety of engineered T-cell immunotherapy, we developed a therapeutic transgene with 4 components: (1) a TCR specific for the hematopoietic-restricted, leukemia-associated minor H antigen, HA-1; (2) a CD8 coreceptor to promote function of the class I-restricted TCR in CD4+ T cells; (3) an inducible caspase 9 safety switch to enable elimination of the HA-1 TCR T cells in case of toxicity; and (4) a CD34-CD20 epitope to facilitate selection of the engineered cell product and tracking of transferred HA-1 TCR T cells. The T-cell product includes HA-1 TCR CD4+ T cells to augment the persistence and function of the HA-1 TCR CD8+ T cells and includes only memory T cells; naive T cells are excluded to limit the potential for alloreactivity mediated by native TCR coexpressed by HA-1 TCR T cells. We describe the development of this unique immunotherapy and demonstrate functional responses to primary leukemia by CD4+ and CD8+ T cells transduced with a lentiviral vector incorporating the HA-1 TCR transgene construct.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Imunoterapia , Leucemia/terapia , Linfócitos T/imunologia , Células Cultivadas , Humanos , Leucemia/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Oligopeptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
BACKGROUND: We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates. METHODS: We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Baclight LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity. RESULTS: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates. CONCLUSIONS: These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells.
Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Minerais/farmacologia , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana , Escherichia coli/fisiologia , Escherichia coli/ultraestrutura , Citometria de Fluxo , Staphylococcus aureus Resistente à Meticilina/fisiologia , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de TransmissãoRESUMO
Proteins created from recurrent fusion genes like CBFB-MYH11 are prevalent in acute myeloid leukemia (AML), often necessary for leukemogenesis, persistent throughout the disease course, and highly leukemia specific, making them attractive neoantigen targets for immunotherapy. A nonameric peptide derived from a prevalent CBFB-MYH11 fusion protein was found to be immunogenic in HLA-B*40:01+ donors. High-avidity CD8+ T cell clones isolated from healthy donors killed CBFB-MYH11+ HLA-B*40:01+ AML cell lines and primary human AML samples in vitro. CBFB-MYH11-specific T cells also controlled CBFB-MYH11+ HLA-B*40:01+ AML in vivo in a patient-derived murine xenograft model. High-avidity CBFB-MYH11 epitope-specific T cell receptors (TCRs) transduced into CD8+ T cells conferred antileukemic activity in vitro. Our data indicate that the CBFB-MYH11 fusion neoantigen is naturally presented on AML blasts and enables T cell recognition and killing of AML. We provide proof of principle for immunologically targeting AML-initiating fusions and demonstrate that targeting neoantigens has clinical relevance even in low-mutational frequency cancers like fusion-driven AML. This work also represents a first critical step toward the development of TCR T cell immunotherapy targeting fusion gene-driven AML.
Assuntos
Leucemia Mieloide Aguda , Animais , Carcinogênese , Subunidade beta de Fator de Ligação ao Core/genética , Humanos , Leucócitos , Camundongos , Mutação , Cadeias Pesadas de Miosina/genética , Proteínas de Fusão Oncogênica/genéticaRESUMO
BACKGROUND: Recent studies have demonstrated that several mineral products sold for medicinal purposes demonstrate antimicrobial activity, but little is known about the physicochemical properties involved in antibacterial activity. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro mineral suspension testing, we have identified two natural mineral mixtures, arbitrarily designated BY07 and CB07, with antibacterial activity against a broad-spectrum of bacterial pathogens. Mineral-derived aqueous leachates also exhibited antibacterial activity, revealing that chemical, not physical, mineral characteristics were responsible for the observed activity. The chemical properties essential for bactericidal activity against Escherichia coli were probed by testing antibacterial activity in the presence of metal chelators, the hydroxyl radical scavenger, thiourea, and varying pH levels. Chelation of the BY07 minerals with EDTA or desferrioxamine eliminated or reduced BY07 toxicity, respectively, suggesting a role of an acid-soluble metal species, particularly Fe(3+) or other sequestered metal cations, in mineral toxicity. This conclusion was supported by NMR relaxation data, which indicated that BY07 and CB07 leachates contained higher concentrations of chemically accessible metal ions than leachates from non-bactericidal mineral samples. CONCLUSIONS/SIGNIFICANCE: We conclude that the acidic environment of the hydrated minerals significantly contributes to antibacterial activity by increasing the availability and toxicity of metal ions. These findings provide impetus for further investigation of the physiological effects of mineral products and their applications in complementary antibacterial therapies.