We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK subsets. CAR.CD19-NK cells display higher anti-leukemic activity towards CD19 + cell lines and primary blasts obtained from patients with B-cell precursor ALL compared to unmodified NK cells. In vivo animal model data showed that the anti-leukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically-modified peripheral blood NK cells for effective 'off-the-shelf' immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.
Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity.Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 medulloblastoma patients. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02+ DAOY cells as well as primary HLA-A*02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T cell-related toxicity, an inducible caspase 9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically-modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating HLA-A*02+ medulloblastoma patients.
Two novel IL2-dependent cell lines, DERL-2 and DERL-7, were established from a patient with hepatosplenic gammadelta T cell lymphoma. This patient presented, at diagnosis, two discrete populations of CD56+ cells, one TCRgammadelta+, the second lacking T cell-restricted antigens. The cell lines derived displayed features corresponding to the two cellular components of the disease: DERL-2 was CD56+/CD3+/TcRgammadelta+ while DERL-7 was CD56+/CD3-/TcRgammadelta-. Along with CD56, the two cell lines shared the expression of CD7, CD2, CD158b and CD117. Karyotype analysis showed that both cell lines were near-diploid, with iso-7q and loss of one chromosome 10. In addition, DERL-2 showed 5q+ in all metaphases analyzed, while DERL-7 revealed loss of one chromosome 4. Genotypically, both cell lines shared the same STR pattern at nine loci and demonstrated an identical rearranged pattern of the T cell receptor genes beta, gamma and delta, with respect to the original tumor cells. These data indicated that both cell lines and the original neoplastic populations were T cell-derived and arose from a common ancestor. Among a large panel of cytokines tested, only SCF was able to substitute IL2 in supporting cell proliferation. Moreover, SCF and IL2 acted synergistically, dramatically enhancing cell growth. These cell lines may represent a model to further analyze the overlap area between T and NK cell malignancies, and may provide new information about the synergistic action of IL2 and SCF on normal and neoplastic T/NK cells.
The costimulatory combination of CD28.OX40 is crucial for improving persistence, in vivo expansion and proliferation of CAR.CD30 T-cells upon tumor encounter. CAR.CD30 manufacturing process based on the use of IL7/IL15 is also of paramount importance to optimize the anti-lymphoma activity of CAR T cells.
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