RESUMEN
The application of immunotherapies such as chimeric antigen receptor (CAR) T therapy or bi-specific T cell engager (BiTE) therapy to manage myeloid malignancies has proven more challenging than for B-cell malignancies. This is attributed to a shortage of leukemia-specific cell-surface antigens that distinguish healthy from malignant myeloid populations, and the inability to manage myeloid depletion unlike B-cell aplasia. Therefore, the development of targeted therapeutics for myeloid malignancies, such as acute myeloid leukemia (AML), requires new approaches. Herein, we developed a ligand-based CAR and secreted bi-specific T cell engager (sBite) to target c-kit using its cognate ligand, stem cell factor (SCF). c-kit is highly expressed on AML blasts and correlates with resistance to chemotherapy and poor prognosis, making it an ideal candidate for which to develop targeted therapeutics. We utilize γδ T cells as a cytotoxic alternative to αß T cells and a transient transfection system as both a safety precaution and switch to remove alloreactive modified cells that may hinder successful transplant. Additionally, the use of γδ T cells permits its use as an allogeneic, off-the-shelf therapeutic. To this end, we show mSCF CAR- and hSCF sBite-modified γδ T cells are proficient in killing c-kit+ AML cell lines and sca-1+ murine bone marrow cells in vitro. In vivo, hSCF sBite-modified γδ T cells moderately extend survival of NSG mice engrafted with disseminated AML, but therapeutic efficacy is limited by lack of γδ T-cell homing to murine bone marrow. Together, these data demonstrate preclinical efficacy and support further investigation of SCF-based γδ T-cell therapeutics for the treatment of myeloid malignancies.
Asunto(s)
Leucemia Mieloide Aguda , Ratones , Animales , Ligandos , Proteínas Tirosina Quinasas Receptoras , Proteínas Proto-Oncogénicas c-kit/genética , Inmunoterapia Adoptiva , Factor de Células MadreRESUMEN
While targeting CD19+ hematologic malignancies with CAR T cell therapy using single chain variable fragments (scFv) has been highly successful, novel strategies for applying CAR T cell therapy with other tumor types are necessary. In the current study, CAR T cells were designed using a ligand binding domain instead of an scFv to target stem-like leukemia cells. Thrombopoietin (TPO), the natural ligand to the myeloproliferative leukemia protein (MPL) receptor, was used as the antigen binding domain to engage MPL expressed on hematopoietic stem cells (HSC) and erythropoietic and megakaryocytic acute myeloid leukemias (AML). TPO-CAR T cells were tested in vitro against AML cell lines with varied MPL expression to test specificity. TPO-CAR T cells were specifically activating and cytotoxic against MPL+ leukemia cell lines. Though the TPO-CAR T cells did not extend survival in vivo, it successfully cleared the MPL+ fraction of leukemia cells. As expected, we also show the TPO-CAR is cytotoxic against MPL expressing bone marrow compartment in AML xenograft models. The data collected demonstrate preclinical potential of TPO-CAR T cells for stem-like leukemia through assessment of targeted killing of MPL+ cells and may facilitate subsequent HSC transplant under reduced intensity conditioning regimens.
Asunto(s)
Leucemia Mieloide Aguda , Trombopoyetina , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Ligandos , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Citocinas , Receptores de Trombopoyetina/genética , Linfocitos T/metabolismo , Trombopoyetina/metabolismoRESUMEN
Hematopoietic stem and progenitor cell (HSPC) lentiviral gene therapy is a promising strategy toward a lifelong cure for hemophilia A (HA). The primary risks associated with this approach center on the requirement for pre-transplantation conditioning necessary to make space for, and provide immune suppression against, stem cells and blood coagulation factor VIII, respectively. Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA alkylation, that increase risk of sterility, infection, and developing secondary malignancies. In the current study, we describe a non-genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies to provide transient immune suppression against the transgene product in a murine HA gene therapy model. This strategy provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor VIII variant, termed ET3. Factor VIII procoagulant activity levels were durably elevated into the normal range and phenotypic correction achieved. Furthermore, no immunological rejection or development of anti-ET3 immunity was observed. These preclinical data support clinical translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for HA.